WO2008033739A2 - Benzimidazole carboxamide derivatives - Google Patents

Benzimidazole carboxamide derivatives Download PDF

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WO2008033739A2
WO2008033739A2 PCT/US2007/077959 US2007077959W WO2008033739A2 WO 2008033739 A2 WO2008033739 A2 WO 2008033739A2 US 2007077959 W US2007077959 W US 2007077959W WO 2008033739 A2 WO2008033739 A2 WO 2008033739A2
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alkyl
compound
hydrate
salt
substituted
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French (fr)
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WO2008033739A3 (en
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Jayaraman Chandrasekhar
Qin Guo
David C. Ihle
Ping Ge
David J. Wustrow
Bertrand L. Chenard
Kevin J. Hodgetts
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Neurogen Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/14—Radicals substituted by nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—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 only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems

Definitions

  • This invention relates generally to benzimidazole carboxamide derivatives, and to the use of such compounds to treat conditions responsive to bradykinin receptor-1 (B]) modulation.
  • the invention further relates to the use of such compounds as reagents for the identification of other agents that bind to Bi, and as probes for the detection and localization of B].
  • Bradykinin is a ⁇ onapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) that functions in cardiovascular homeostasis, contraction and relaxation of smooth muscles, inflammation and pain.
  • BK Bradykinin
  • the effects of BK are mediated by specific G protein-coupled BK receptors, of which there are at least two distinct subtypes termed Bi and B 2 .
  • the B 2 receptor is expressed constitutively in a variety of tissues.
  • the Bi receptor is inducibly expressed in response to pathophysiological conditions such as inflammation, pain, trauma, bacterial infection, burns and shock.
  • Bj is a particularly attractive drug target for these and other conditions, and agents that act at this receptor may be targeted specifically to injured tissues, with minimal effects in normal tissues.
  • compounds that bind to B 1 and/or modulate the activity of Bj also find use as research tools.
  • the present invention provides benzimidazole carboxamide derivatives that satisfy Formula I:
  • A, B, D and E are each CH or N; preferably such that no more than one of A, B, D and E is N;
  • Y is a group of the formula (CH 2 ) I -Q-(CH 2 ) P , which is optionally substituted and is preferably substituted with from 0 to 4 substituents independently chosen from (i) amino, hydroxy, cyano, C]-C 6 alkyl, C 2 -C 6 aikenyl, C 3 -Cc,alkynyl and C 1 -C ⁇ haloalkyl; and (ii) substituents of the same carbon atom or adjacent carbon atoms that are taken together to form C 3 -C 6 cycioalkyl, wherein:
  • R 1 represents from 0 to 4 substituents; preferably each substituent is independently chosen from halogen, hydroxy, cyano, amino, nitro, aminocarbonyl, aminosulfonyl, -COOH, C 1 -C 6 a!kyl, C 2 - Qalkenyl, C 2 -C 6 alkynyl, (C 3 -C 3 cyc!oa!kyl)Co-C 4 a!kyl, C 1 -C 6 alkoxy ; C 1 -C 6 alky!thio, C,-
  • R 2 is C 1 -C 6 alkyl, C r C 6 alkenyl, C 2 -C 6 a]kynyl, (C 3 -C 8 cycloalkyI)C 0 -C 4 alkyl, mono- or di-(C,-
  • R 3 is hydrogen, CrQalkyl or taken together with R i0 to form a 4- to 7-membered optionally substituted heterocycloaikyl; and R 4 and R 5 are: (i) independently chosen from C 1 -C 6 alkyl, C 2 -C6alkenyl, C 2 -Qalkynyl, (C3-C 3 cycIoalkyI)Co- C 4 alkyl, (4- to 7-rnembered heterocycloaIkyl)C 0 -C 4 aikyl, phenylC 0 -C 4 alkyl and (5- to 10- membered heteroaiyl)Co-C 4 aIkyl; or
  • benzimidazole carboxamide derivatives of Formula I, and other Formulas provided herein are B 1 modulators and exhibit a K 1 of no greater than 5 micromolar, 2 micromolar, 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar or 10 nanomolar in a Bi binding assay and/or have an EC 50 or IC 50 value of no greater than 5 micromolar, 2 micromolar, 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar or 10 nanomolar in an assay for determination of Bi agonist or antagonist activity.
  • B] modulators provided herein are Bi antagonists; preferably such antagonists exhibit no detectable Bj agonist activity.
  • benzimidazole carboxamide derivatives of Formula 1 are labeled with a detectable marker (e.g., radiolabeled or fluorescein conjugated).
  • a detectable marker e.g., radiolabeled or fluorescein conjugated
  • the present invention further provides, within other aspects, pharmaceutical compositions comprising at least one benzimidazole carboxamide derivative of Formula ⁇ in combination with a physiologically acceptable carrier or excipient.
  • the present invention further provides, within other aspects, pharmaceutical compositions comprising at least one benzimidazole carboxamide derivative as described herein in combination with a physiologically acceptable carrier or excipient.
  • Methods are further provided for inhibiting agonist-induced B 1 activity.
  • the inhibition takes place in vitro.
  • Such methods comprise contacting a B 1 receptor with at least one Bj antagonist as described herein, under conditions and in an amount or concentration sufficient to detectably inhibit agonist-induced B] activity.
  • the Bi receptor is in a patient.
  • Such methods comprise contacting ceils expressing a B] receptor in a patient with at least one B] antagonist as described herein in an amount or concentration that would be sufficient to detectabiy inhibit agonist-induced Bj activity in cells expressing a cloned B, receptor in vitro.
  • the present invention further provides methods for treating a condition responsive to Bj receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of at least one benzimidazole carboxamide derivative of Formula 1.
  • methods for treating pain in a patient, comprising administering to a patient suffering from (or at risk for) pain a therapeutically effective amount of at least one benzimidazole carboxamide derivative of Formula I.
  • Pain conditions that may be treated include, but are not limited to, inflammatory pain, acute pain, dental pain, back pain, surgical pain, headache, neuropathic pain, and pain associated with osteoarthritis or trauma.
  • the present invention provides methods for determining the presence or absence of Bi in a sample, comprising: (a) contacting a sample with a benzimidazole carboxamide derivative of Formula 1 under conditions that permit binding of the compound to Bj; and (b) detecting a signal indicative of a level of the compound bound to Bj.
  • the present invention provides methods of preparing the compounds disclosed herein, including the intermediates.
  • the present invention provides benzimidazole carboxamide derivatives, which may be used in vitro or in vivo in a variety of contexts, as described herein.
  • TERMINOLOGY 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. 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. If 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. Certain compounds are described herein using a general formula that includes variables ⁇ e.g., Y, R]).
  • each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a fo ⁇ nula is defined independently at each occurrence.
  • the term "benzimidazole carboxamide derivative” encompasses any compound that satisfies Formula I. This term further includes pharmaceutically acceptable salts, solvates (e.g., hydrates) and esters of such compounds.
  • a "pharmaceutically acceptable salt” of a compound recited herein is an acid or base salt that is 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.
  • Specific pharmaceutically acceptable anions for use in salt formation include, but are not limited to.
  • salt formation examples include, but are not limited to ammonium, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, and metais such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.
  • pharmaceutically acceptable salts for the compounds provided herein include, but are not limited to ammonium, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, and metais such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.
  • 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.
  • 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, methanol, isopropanol or acetonitrile, is preferred.
  • nonaqueous media such as ether, ethyl acetate, ethanol, methanol, isopropanol or acetonitrile, is preferred.
  • each compound provided herein may, but need not. be formulated as a solvate (e.g., a hydrate) or non-covalcnt complex.
  • prodrugs of the compounds provided herein are a compound that may not fully satisfy the structural requirements of a formula provided herein, but is modified in vivo, following administration to a patient, to produce a compound within the scope of such formula.
  • a prodrug may be an acylated derivative of a compound as provided herein.
  • Prodrugs include compounds wherein hydroxy, amine or suifhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, amino, or sulfhydryl group, respectively.
  • prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein.
  • Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to yield the parent compounds.
  • alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon.
  • Alkyl groups include groups having from 1 to 8 carbon atoms (CpCgalkyl), from 1 to 6 carbon atoms (CpQalkyl) and from 1 to 4 carbon atoms (C 1 -C 4 alkyI), such as methyl, ethyl, propyl, isopropyl, n-buty ⁇ , sec-butyl, fer/-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl or 3-methyipentyI.
  • C 0 -C 4 alkyl refers to a single covalent bond (C 0 ) or an alkylene group having 1, 2, 3 or 4 carbon atoms; "C 0 -C 2 alky” refers to a single covalent bond or a methylene or ethylene group.
  • Alkylene refers to a divalent alkyl group, as defined above.
  • C 1 -C 4 alkylene is an alkylene group having 1, 2. 3 or 4 carbon atoms.
  • Alkenyl refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C 2 -C 8 alkenyl, C 1 -Cgalkenyl and C 2 - C 4 alkenyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively, such as ethenyl, ally! or isopropenyl.
  • Alkynyl refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond.
  • Alkynyl groups include C 1 -C 8 alkynyl, C 2 -C 6 alkynyl and C 2 -C 4 a!kynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.
  • a “cycloalkyl” is a saturated or partially saturated cyclic group in which all ring members are carbon, such as cyclopropyi, cyclobutyl, cyclopentyl, cyclohexyl and partially saturated variants thereof. Certain cycloalkyl groups are C 3 -C 8 cycloalkyl, in which the ring contains from 3 to 8 ring members, all of which are carbon.
  • a "( C 3 -C 8 cycloalkyl)C 0 -C 4 alkyl” is a C 3 -C 8 cycloalkyi group linked via a single covalent bond or a C 1 -C 4 alkylene group.
  • alkoxy is meant an alkyl group attached via an oxygen bridge (i.e., -O- alkyl).
  • Alkoxy groups include C 1 -C ⁇ alkoxy and C 1 -C 4 alkoxy groups, which have from 1 to 6 or from
  • 3-methyI ⁇ entoxy are representative alkoxy groups.
  • alkylthio refers to an alkyl group as attached via a sulfur bridge (i.e., -S-alkyl).
  • Alkylthio groups include C 1 -C 6 alkylthio and C 1 -C 4 aikylthio groups, which have from 1 to 6 or from I to 4 carbon atoms, respectively.
  • Alkylsulfmyl refers to groups of the formula -(SO)-alkyl, in which the sulfur atom is the point of attachment.
  • Alkytsulfinyl groups include C 1 -C 6 alkylsulfmyl and C 1 -C 4 alkylsulf ⁇ nyl groups, which have from I to 6 or from 1 to 4 carbon atoms, respectively.
  • Alkyisulfonyj refers to groups of the formula - ⁇ SO 2 )-aIkyL in which the sulfur atom is the point of attachment.
  • Alkylsulfonyl groups include C 1 -C ⁇ alkylsulfonyl and C 1 -Qalkylsulfony! groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively.
  • C 4 alkyl refers to a C 1 -C 6 alkylsulfonyl that is linked via a single covale ⁇ t bond or a C 1 -C 4 alkylene group (i.e., -(C 0 -C 4 alkylHSO 2 MC 1 -C 6 alkyl)).
  • Alkoxycarbonyl groups include C]-Cg, Q-C ⁇ and Q- C 4 alkoxycarbonyl groups, which have from 1 to 8, 6 or 4 carbon atoms, respectively, in the alkyl portion of the group.
  • Alkylamino refers to a secondary or tertiary amine that has the general structure -NH-alkyl or -N(alkyi)(alkyl), wherein each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups.
  • groups include, for example, mono- and di-(C 1 -C s alkyi)amino groups, in which each C 1 -C s alkyl may be the same or different, as well as mono- and di ⁇ (C 1 -Q,alkyl)amino groups and mono- and di-(C 1 -C 4 alkyl)amino groups.
  • Alkylaminoalkyl refers to an alkylamino group linked via an alkylene moiety (Le , a group having the general structure -aikyiene-NH-alkyl or -alkylene-N(alkyl)(alkyl)) in which each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups.
  • Alkylaminoalkyl groups include, for example, mono- and di-(C;-CsaIkyl)aminoCrC 6 alkyl, and mono- and di-(C 1 - C 6 aIkyI)aminoC 1 -C 1 alkyl.
  • “Mono- or di-(C 1 -C 6 aIky])aminoCo-C 4 aIkyl” refers to a mono- or di-(C t - C 6 alkyl)amino group linked via a single covalent bond or a C 1 -C 4 alkylene group.
  • alkyl as used in the terms “alkylamino” and “alkylaminoalkyl” differs from the definition of "alkyl” used for all other alkyi-containing groups, in the inclusion of cycloalkyl and (cyclnalkyl)a ⁇ cyl groups (e g , (CrC ⁇ cyrlnaIkyl)Co-C 2 a!kyl)
  • aminonosulfonyl refers Io a sulfonamide group (i. e , -SO 2 NH 2 ).
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • haloalkyl is an aikyl group that is substituted with 1 or more independently chosen halogens (e.g., "C 1 -C 8 haloalkyl” groups have from 1 to 8 carbon atoms: "C 1 -C 6 haioalkj l” groups have from i to 6 carbon atoms).
  • haloaikyi groups include, but are not limited to, mono-, di- or tri-fluoromethy ⁇ ; mono-, di- or tri-chlororaethyl; mono-, di-, tri-.
  • Typical haloalkyl groups are trifluoromethy] and difluoromethyl.
  • a dash (“-") that is not between two letters or numbers is used to indicate a point of attachment for a substituent.
  • Phenyl groups linked via a single covalent bond or Q-dalkylene group are designated pheny!C 0 -C 4 alkyl (e.g., phenylC,-C 2 alkyI, which includes benzyl, l ⁇ henyl-ethyl and 2-phenyl-ethyl). When substituted, it will be apparent that such groups may be substituted on the ring portion and/or on the alkylene portion of the group.
  • a “heterocycle” (also referred to herein as a “heterocyclic group”) has from 1 to 3 fused, pendant or spiro rings, at least one of which is a heterocyclic ring (i.e., one or more ring atoms is a heteroatom independently chosen from oxygen, sulfur and nitrogen, with the remaining ring atoms being carbon).
  • a heterocyclic ring comprises 1, 2, 3 or 4 heteroatoms; within certain embodiments each heterocyclic ring has 1 or 2 heteroatoms per ring.
  • Each heterocyclic ring generally contains from 3 to 8 ring members (rings having from 4 or 5 to 7 ring members are recited in certain embodiments) and heterocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members.
  • Certain heterocycles comprise a sulfur atom as a ring member; in certain embodiments, the sulfur atom is oxidized to SO or SC ⁇ - Heterocycles may be optionally substituted with a variety of substituents, as indicated.
  • Certain heterocycles are 4- to 10-membered and comprise one or two rings; in certain embodiments, such heterocycles are monocyclic (e.g., 4- to 8-membered, 5- to 8-membered, 4- to 7-membered, or 5- or 6-membered).
  • heterocycles are heteroaryi groups (i.e., at least one heterocyclic ring within the group is aromatic), such as a 5- to 10-membered heteroaryi (which may be monocyclic or bicyclic) or a 5- or
  • 6-membered heteroaryi e.g., tbtenyl, imidazolyl, pyridyl or pyrimidyl.
  • Other heterocycles are heterocycloalky] groups (i.e., do not comprise an aromatic heterocyclic ring).
  • Certain heterocycies may be linked by a single covalent bond or via an alkylene group, as indicated, for example, by the term "(4- to 7-m ⁇ mber ⁇ d "(4- to 7-membered heterocycloalky ⁇ )C 1 -
  • C 2 alkyl refers to a 4- to 7-membered heterocycloalkyl that is linked via a methylene or ethylene linker. When substituted, it will be apparent that substituent(s) may be attached on the ring and/or the alkylene linker.
  • a “substituent,” as used herein, refers to a molecular moiety that is covIERly 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 group discussed herein that is covendingly bonded to an atom (such as a carbon or nitrogen atom) that is a ring member.
  • substitution refers to replacing a hydrogen atom in a molecular structure with a substituent as described above, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
  • Groups that are "optionally substituted' 1 are unsubstituted or are substituted by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different).
  • Optional substitution is also indicated by the phrase “substituted with from 0 to X substituents," where X is the maximum number of possible substituents.
  • Certain optionally substituted groups are substituted with from 0 to 2, 3 or 4 independently selected substituents (i.e , are unsubstituted or substituted with up to the recited maximum number of substituents).
  • B 1 refers to the human B 1 bradykinin receptor reported by Menke ei al.
  • agonist refers to a compound that binds B 1 and induces signal transduction mediated by B 1 .
  • Bi agonists include, for example, bradykinin and kallidin (lysyl-bradykinin), as well as peptide portions or variants of bradykinin or kallidin that bind B; and retain activity.
  • Representative B s agonists include, but are not limited to. desArg 9 bradykinin and desArg 10 kallidin.
  • a "B] antagonist” is a compound that detectably inhibits signal transduction mediated by Bj. Such inhibition may be determined using the representative calcium mobilization assay provided in Example 7.
  • Preferred Bi antagonists have an IC 50 Of 5 ⁇ M or less in this assay, more preferably 2 ⁇ M or less, and stili more preferably 1 ⁇ M or less, 500 nM or less, 100 iiM or less or 10 nM or less.
  • the B) antagonist is specific for Bi (i.e., the 3C 50 value in a similar assay performed using the Bj receptor is greater than 2 ⁇ M and/or the IC 50 ratio (B7/B 1 ) is at least 10, preferably 100, and more preferably at least 1000).
  • Bj antagonists preferably have minimal agonist activity (i.e., induce an increase in the basal activity of Bj that is less than 5% of the increase that would be induced by one EC 50 of the peptide agonist desArg'°kaliidin, and more preferably have no detectable agonist activity within the assay described in Example 7).
  • B 1 antagonists for use as described herein are generally non-toxic.
  • B; antagonists include neutral antagonists and inverse agonists.
  • a "neutral antagonist" of B 1 is a compound that inhibits the activity of B 1 agonist (e.g , desArg 10 kaIlidin) at Bj, but does not significantly change the basal activity of the receptor (i.e., within a calcium mobilization assay as described in Example 7 performed in the absence of agonist.
  • Bi activity is reduced by no more than 10%, more preferably by no more than 5%. and even more preferably by no more than 2%; most preferably, there is no detectable reduction in activity).
  • Neutral antagonists may. but need not. also inhibit the binding of agonist to Bi.
  • An "inverse agonist" of Bi is a compound that reduces the activity of Bi below its basal activity level in the absence of actuating concentrations of agonist.
  • Inverse agonists may also inhibit the activity of agonist at Bi, and/or may inhibit binding of Bj agonist to Bi.
  • the reduction in basal activity of B, produced by an inverse agonist may be determined from a calcium mobilization assay, such as the assay of Example 7.
  • a “therapeutically effective amount” is an amount that, upon administration to a patient, results in a discernible patient benefit (e.g., provides detectable relief from a condition being treated). Such relief may be detected using any appropriate criteria.
  • a therapeutically effective amount or dose generally results in a concentration of compound in a body fluid (such as blood, piasma, serum, CSF, synovial fluid, lymph, cellular interstitial fluid, tears or urine) that is sufficient to result in detectable alteration in B r mediated signal transduction (using an assay provided herein).
  • the discernible patient benefit may be apparent after administration of a single dose, or may become apparent following repeated administration of the therapeutically effective dose according to a predetermined regimen, depending upon the indication for which the compound is administered.
  • a "patient” is any individual treated with a benzimidazole carboxamide derivative as provided herein. Patients include humans, as well as other animals such as companion animals (e.g., dogs and cats) and livestock. Patients may be experiencing one or more symptoms of a condition responsive to Bi modulation or may be free of such symptom(s) (i.e., treatment may be prophylactic in a patient considered to be at risk for the development of such symptoms).
  • the present invention provides benzimidazole carboxamide derivatives of Formula I that may be used in a variety of contexts, including in the treatment of conditions responsive to Bj modulation, as described herein.
  • Such compounds may also be used within in vitro assays (e.g., assays for Bi activity), as probes for detection and localization of B, and within assays to identify other B 1 antagonists.
  • Ri represents from 0 to 2 substituents independently chosen from halogen, cyano and C 1 -C ⁇ alkyl.
  • variable represented by R 2 is, within certain embodiments, phenylQ-C ⁇ alky] that is substituted with from 0 to 3 substituents independently chosen from halogen, hydroxy, cyano, Q- Qaikyl, and Q-Qalkoxy.
  • R 2 moieties include, for example, benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, Q-C 4 alkyl, and C 1 - C 4 alkoxy.
  • the variable represented by R 2 is (4- to 7-membered heterocyc!oa!kyl)Q-C 1 aikyf that is substituted with from 0 to 3 substituents independently chosen from 0x0 and Q-C 4 alkyl.
  • Y the sum of r and p ranges from 1 to 6 for certain benzimidazole carboxamide derivatives. Within certain such compounds, p is not zero.
  • Certain Y groups satisfy the formula (CH 2 )rO-(CH 2 ) p , which is substituted with from 0 to 4 substituents independently chosen from amino, hydroxy, cyano, Q-Cealkyl, Ci-Cealkenyl. C 2 -C 6 alkynyl, Q- C ⁇ haloalkyl and substituents of the same carbon atom or adjacent carbon atoms that are taken together to form CrC f ,cycloaIkyl.
  • Y groups include, for example, -O-CH 2 -, -CH 2 -O- CH 2 - -0-CH 2 -CH 2 - -CH 2 -O-CH 2 -CH 2 - and -CH 2 -CH 2 -O-CH 2 -.
  • Other Y groups satisfy the formula -N(R 10 )-CH 2 -.
  • Y is a group of the formula (CH 2 V(CH 2 ),, that is substituted with from 0 to 4 substituents independently chosen from amino, hydroxy, cyano, C 1 -C ⁇ alkyl.
  • Y groups include, for example, Q-Qalkylene groups such as methylene and ethylene, each of which is optionally substituted as indicated above (e.g., unsubstituted or substituted with amino, hydroxy, cyano, Q -Chalky! (e.g., methyl or ethyl), C 2 -C 4 alkenyl or Q-dhaloalkyl).
  • Q-Qalkylene groups such as methylene and ethylene, each of which is optionally substituted as indicated above (e.g., unsubstituted or substituted with amino, hydroxy, cyano, Q -Chalky! (e.g., methyl or ethyl), C 2 -C 4 alkenyl or Q-dhaloalkyl).
  • R 1 is hydrogen or Q-Qalkyl.
  • Y is a group of the formula (CHJrCHR m -. wherein R iU is taken together with Rj to form a 4- to 7-rncrnbcrcd heterocycloalkyl. Certain such compounds further satisfy Formula II or Ha:
  • the variables R 4 and K 5 are taken together to form a 4- to 7-membered heterocycloafkyl that is substituted with mono- or di-(CrC 6 alkyi)aminoCo-C 4 alkyl, (4- to 7-membered heterocycloa!kyl)Co-C ⁇ alkyl, ⁇ heny[C 0 -C 4 alkyl or (6-membered heteroaryl)Co-C 4 aIkyl, each of which is unsubstituted or substituted with oxo, C,- C 4 alkyl or (5- or 6-membered heterocycIe)Co-C 2 alkyI. Certain such compounds further satisfy Formula III or Ilia:
  • R 6 is piperidinylQrGtalkyl, pyrro ⁇ idinylCo-Qaikyl, pyridmylC 0 -C 4 alkyl, pyrimidinyiC 0 -C 4 aIkyl, trtrahydropyrimidinylCo ⁇ alkyl, imidazolylCo-C 4 alkyl, dihydroimidazoiylCo- C 4 alkyl, thiazolylCo-C 4 alkyl, oxadiazolylCo-C 4 alkyl, phenyiCo-C 4 alkyI, or di-(C 1 -C 4 alkyl)aminoC 0 - C 4 alkyl, each of which is unsubstituted or substituted with one or two C 1 -C 4 alkyl.
  • R 6 is piperidinylQrGtalkyl, pyrro ⁇ idinylCo-Qaikyl, pyridmylC 0
  • R] represents from 0 to 2 substituents independently chosen from halogen, cyano, and C 1 -C 4 alkyl;
  • R. 2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen,
  • R 3 is hydrogen or C 1 ⁇ alkyl
  • Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
  • R 4 is CrC 4 alkyl; and R 5 is phenylCo-C 2 aIkyl that is substituted with mono- or di-(C 1 -C 6 alkyI)aminoC 0 -C 4 aIkyl, (4- to 7- membered heterocycloalkyl)C 0 -C 4 alkyl, phenylC 0 -C 4 alkyl or (6-membered heteroaryl)C 0 -C 4 aIkyl, each of which is unsubstituted or substituted with oxo, C 1 -C 4 alkyl, (5- or 6-membered heteiOcycle)C 0 - C ⁇ alkyl, or mono- or di-(C]-Q,alky!aminoCo-Qaikyl. Certain such compounds further satisfy Formula IV or IVa:
  • R 7 is methyl or ethyl; and R s is pi ⁇ eridinylC 0 -C 4 aIkyl, pyrrolidiny[C[rC 4 alkyI, pyridinylC 0 - C 4 alkyl, pyrimidinylCo-C 4 aIky[, trtrahydropyrimidinylCo-C ⁇ alkyl.
  • represents from 0 to 2 substituents independently chosen from halogen, cyano. and C 1 -C 4 aIkyl;
  • R 2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, C 1 -C 4 alkyl, and C]-C 4 alkoxy;
  • R 3 is hydrogen or CrQalkyl; and
  • Y is methylene or ethylene, each of which is un substituted or substituted with methyl or ethyl.
  • R 4 and R 5 are taken together to form a 8- to 14-membered spiro heterocycloaikyl that is unsubstituted or substituted with C 1 -C 4 alkyl, (C 1 -C 7 cyc]oalkyl)C 0 -C 2 alkyl or (5- or 6-membered heterocycle)C 0 "C 2 alkyL
  • Certain such compounds further satisfy Formula V or Va:
  • n is independently 0 or 1 ;
  • X is O or NR 9 ; and
  • R 9 is CrC 4 a!kyl or (C 3 -C 7 cyc ⁇ oalkyl)Co- C 2 alkyl.
  • Ri represents from 0 Io 2 substituents independently chosen from halogen, cyano. and CrC 4 alkyl; R 2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen,
  • R 3 is hydrogen or C]-C 4 a]ky ⁇
  • Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
  • benzimidazole carboxamide derivatives provided herein include, but are not limited to, those specifically described in the Examples below. It will be apparent that the specific compounds recited herein are representative only, and are not intended to limit the scope of the present invention. Fuilher, as noted above, all compounds of the present invention may be present as a free acid or base or as a pharmaceutically acceptable salt, hydrate or ester.
  • benzimidazole carboxamide derivatives provided herein are Bj modulators.
  • certain benzimidazole carboxamide derivatives provided herein are specific for B] .
  • B) modulator activity may be confirmed using a calcium mobilization a ⁇ sa v such as the assa ⁇ described in EXiim n ls 7 herein
  • B 1 binding activity of the benzimidazole carboxamide derivatives provided herein may be confirmed using the representative assay described in Example 6, herein, or using an assay described by Fox et al. (2005) Br. J. Pharmacol. 144:889-99.
  • Preferred B, modulators exhibit a K 1 within such an assay of 5 micromolar or less, more preferably 2 micromolar or less, 1 rnicromolar or less, 500 nanomolar or less, 100 nanomolar or less or 10 nanomolar or less.
  • B] modulators provided herein may be confirmed using any of a variety of animal models including, but not limited to, those described in the following documents (each of which is hereby incorporated by reference for its disclosure of the recited animal model):
  • compounds provided herein may be evaluated for certain pharmacological properties including, but not limited to, oral bioavailability (preferred compounds are orally bioavailabie to an extent allowing for therapeutically effective doses of less than 140 mg/kg, preferably less than 50 mg/kg, more preferably less than 30 mg/kg, even more preferably less than 10 mg/kg, still more preferably less than 1 mg/kg and most preferably less than 0.1 mg/kg), toxicity (a preferred compound is nontoxic when a therapeutically effective amount is administered to a subject), side effects (a preferred compound produces side effects comparable Io placebo when a therapeutically effective amount of the compound is administered to a subject), serum protein binding and in vitro and in vivo half-fife (a preferred compound exhibits an in vivo half-life allowing for Q.3.
  • oral bioavailability preferred compounds are orally bioavailabie to an extent allowing for therapeutically effective doses of less than 140 mg/kg, preferably less than 50 mg/kg, more preferably less than 30 mg/kg,
  • D. dosing preferably T. I. D, dosing, more preferably B. LD. dosing, and most preferably o ⁇ ce-a- day dosing).
  • differential penetration of the blood brain barrier may be desirable. Routine assays that are well known in the art may be used to assess these properties, and identify superior corn pounds for a particular use. For example, assays used to predict bioavailability include transport across human intestinal cell monolayers, including Caco-2 cell monolayers. Penetration of the blood brain barrier of a compound in humans may be predicted from the brain levels of the compound in laboratory animals given the compound (e.g., intravenously). Serum protein binding may be predicted from albumin binding assays. Compound half-life is inversely proportional to the frequency of dosage of a compound. In vitro half-lives of compounds may be predicted from assays of microsomal half-life as described herein.
  • nontoxic compounds are nontoxic.
  • the term "nontoxic” as used herein shall be understood in a relative sense and is intended to refer to any substance that has been approved by the United States Food and Drug Administration (“FDA”) for administration to mammals (preferably humans) or, in keeping with established criteria, is susceptible to approval by the FDA for administration to mammals (preferably humans).
  • FDA United States Food and Drug Administration
  • a highly preferred nontoxic compound generaily satisfies one or more of the following criteria: (1 ) does not substantially inhibit cellular ATP production; (2) does not significantly prolong heart QT intervals; (3) does not cause substantial liver enlargement, or (4) does not cause substantia! release of liver enzymes.
  • a compound that does not substantially inhibit cellular ATP production is a compound that satisfies the criteria set forth in Example 8, herein.
  • cells treated as described in Example 8 with 100 ⁇ M of such a compound exhibit ATP levels that are at least 50% of the ATP levels detected in untreated cells.
  • such cells exhibit ATP levels that are at least 80% of the ATP levels detected in untreated cells.
  • a compound that does not significantly prolong heart QT intervals is a compound that does not result in a statistically significant prolongation of heart QT intervals (as determined by electrocardiography) in guinea pigs, minipigs or dogs upon administration of a dose that yields a serum concentration equal to the EC 50 or IC 10 for the compound.
  • a dose of 0.01 , 0.05, 0.1 , 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally does not result in a statistically significant prolongation of heart QT intervals.
  • statically significant is meant results varying from control at the p ⁇ 0.I level or more preferably at the p ⁇ 0.05 level of significance as measured using a standard parametric assay of statistical significance such as a student's T test.
  • a compound does not cause substantial liver enlargement if daily treatment of laboratory rodents (e.g., mice or rats) for 5-10 days with a dose that yields a serum concentration equal to the
  • EC 50 or IC 50 for the compound results in an increase in liver to body weight ratio that is no more than
  • such doses do not cause liver enlargement of more than 75% or 50% over matched controls. If non-rodent mammals (e.g., dogs) are used, such doses should not result in an increase of liver to body weight ratio of more than 50%. preferably not more than 25%, and more preferably not more than 10% over matched untreated controls.
  • Preferred doses within such assays include 0.01. 0.05. 0.1. 0.5. 1. 5. 10, 40 or 50 mg/kg ⁇ u ⁇ iiiiistei ed p ⁇ iciitei a ⁇ l ⁇ Oi orally.
  • a compound does not promote substantial release of liver enzymes if administration of twice the minimum dose that yields a serum concentration equal to the EC 50 or 1CTM for the compound does not elevate serum levels of ALT, LDH or AST in laboratory rodents by more than
  • a compound does not promote substantial release of liver enzymes if, in an in vilr ⁇ hepatocyte assay, concentrations (in culture media or other such solutions that are contacted and incubated with hepatocytes in vitro) that are equal to the EC50 or IC 50 for the compound do not cause detectable release of any of such liver enzymes into culture medium above baseline levels seen in media from matched mock-treated controi cells. In more highly preferred embodiments, there is no detectable release of any of such liver enzymes into culture medium above baseline levels when such compound concentrations are five-fold, and preferably ten-fold the EC 5 0 or IC 5 0 for the compound.
  • certain preferred compounds do not inhibit or induce microsomal cytochrome P450 enzyme activities, such as CYP 1A2 activity.
  • CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a concentration equal to the EC 50 or IC 50 for the compound.
  • Certain preferred compounds are not clastogenic (e.g., as determined using a mouse erythrocyte precursor cell micronucleus assay, an Ames raicronucleus assay, a spira! micronucleus assay or the like) at a concentration equal the EC 50 or IC 50 for the compound.
  • certain preferred compounds do not induce sister chromatid exchange (e.g., in Chinese hamster ovary cells) at such concentrations.
  • compounds provided herein may be isotopically-Iabeled or radiolabeled.
  • such compounds may have one or more atoms replaced by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be present in the compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 11 C, ' 3 C, ' 4 C, ' 5 N, 18 O, 17 O 5 31 P, ' 2 P, ' 5 S, 18 F and 36 Cl.
  • substitution with heavy isotopes such as deuterium (i.e., 2 H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.
  • a compound provided herein may contain one or more asymmetric carbon atoms, so that the compound can exist in different stereoisomers forms.
  • Such forms can be, for example, racemates or optically active forms.
  • All stereoisomers are encompassed by the present invention. Nonetheless, it may be desirable to obtain single enantiomers (i.e., optically active forms).
  • Standard methods for preparing single enantiomers include asymmetric synthesis and 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.
  • Each radioisotope is preferably carbon (e.g., 14 C), hydrogen (e.g., 3 I-I), sulfur (e.g., 33 S) or iodine (e.g., 125 I).
  • Tritium labeled compounds may also be prepared catalytically via platinum-catalyzed exchange in tritiated acetic acid, acid-catalyzed exchange in tritiated trifluoroacetic acid, or heterogeneous-catalyzed exchange with tritium gas using the compound as substrate, in addition, certain precursors may be subjected to tritium-halogen exchange with tritium gas, tritium gas reduction of unsaturated bonds, or reduction using sodium borotritide, as appropriate. Preparation of radiolabeled compounds may be conveniently performed by a radioisotope supplier specializing in custom synthesis of radiolabeled probe compounds.
  • compositions comprising one or more benzimidazole carboxamide derivatives provided herein, together with at least one physiologically acceptable carrier or excipient.
  • Pharmaceutical compositions may comprise, for example, one or more of water, buffers (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil. vegetable oil, dimethylsulfoxide, carbohydrates (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.
  • other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein.
  • compositions may be formulated for any appropriate manner of administration, including, for example, topical, oral (including, but not limited to, sublingual), nasal, rectal or parenteral administration.
  • parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar injection or infusion technique.
  • compositions suitable for oral use are preferred.
  • Such compositions include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions of the present invention may be formulated as a lyophilizate.
  • 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 (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin or acacia) and lubricating agents (e.g., magnesium stearate, stearic acid or talc).
  • inert diluents e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents e.g., corn starch or
  • the tablets may be un coated 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 (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 (e.g., peanut oil, liquid paraffin or olive oil).
  • an inert solid diluent e.g., calcium carbonate, calcium phosphate or kaolin
  • an oil medium e.g., peanut oil, liquid paraffin or olive oil
  • Aqueous suspensions coiitd.ni the sctive iiidtertiil(s) in admixture with excipienls suitable foi the manufacture of aqueous suspensions.
  • excipients include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanlh and gum acacia); and dispersing or wetting agents (e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as pol>oxyethylene stearate.
  • Aqueous suspensions may also comprise one or more preservatives, such as 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 ingredient(s) in a vegetable oil ⁇ e.g., arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations.
  • Such suspensions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent e.g., glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerin, glycerin, glycerin, glycerin, glycerin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol, glycerol
  • compositions may also be formulated as oil-in-water emulsions.
  • the oily phase may be a vegetable oil (e.g., olive oil or arachis oil), a mineral oil ⁇ e.g., liquid paraffin) or a mixture thereof.
  • Suitable emulsifying agents include naturally-occurring gums (e.g., gum acacia or gum tragacanth).
  • An emulsion may also comprise one or more sweetening and/or flavoring agents.
  • phosphatides e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol
  • anhydrides e.g., sorbitan monoleate
  • condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide 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.
  • 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 (e.g., ethanol or isopropyl alcohol) or glycerin; glycols (e.g., butylene, isoprene or propylene glycol); aliphatic alcohols (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, acylgiycerols (including oils, such as 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 (e.g., ethanol or isopropyl alcohol) or glycerin
  • glycols e.g., butylene, isoprene
  • 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 and emulsions.
  • 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.
  • a pharmaceutical composition may be prepared as a sterile injectible aqueous or oleaginous suspension.
  • the compound(s) provided herein, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • Such a composition may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents such as those mentioned above.
  • suitable dispersing, wetting and/or suspending agents such as those mentioned above.
  • the acceptable vehicles and solvents that may be employed are water, 1 ,3-butanediol,
  • sterile, fixed oils may be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed, including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectible compositions, and adjuvants such as local anesthetics, preservatives and/or buffering agents can be dissolved in the vehicle.
  • compositions may also be formulated as suppositories (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 may be formulated for release at a pre-detcrmined rate.
  • iiiMdiitdiieuus i elcdSe ificty oe dL ⁇ ievcu foi example, vid
  • Sublingual administration i.e., administration by mouth in such a way that the active ingredient(s) are rapidly absorbed via the blood vessels under the tongue rather than via the digestive tract.
  • Controlled release formulations (/ e., formulations such as a capsule, tablet or coated tablet that slows and/or delays release of active ingredient(s) following administration) may be administered by. for example, oral, rectal or subcutaneous implantation, or by implantation at a target site.
  • a controlled release formulation comprises a matrix and/or coating that delays disintegration and absorption in the gastrointestinal tract (or implantation site) and thereby provides a delayed action or a sustained action over a longer period.
  • One type of controlled-release formulation is a sustained-release formulation, in which at least one active ingredient is continuously released over a period of time at a constant rate.
  • the therapeutic agent is released at such a rate that blood (e g., plasma) concentrations are maintained within the therapeutic range, but below toxic levels, over a period of time that is at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours.
  • 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.
  • Controlled release may be achieved by combining the active ingredient(s) with a matrix material that itself alters release rate and/or through the use of a controlled-release coating.
  • the release rate can be varied using methods well known in the ait, including (a) varying the thickness or composition of coating, (b) altering the amount or manner of addition of plasticizer in a coating, (c) including additional ingredients, such as release-modifying agents, (d) altering the composition, particle size or particle shape of the matrix, and (e) providing one or more passageways through the coating.
  • the amount of modulator contained within a sustained release formulation depends upon, for example, the method of administration (e.g., the site of implantation), the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • the matrix material which itself may or may not serve a controlled-release function, is generally any material that supports the active ingredient(s).
  • a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
  • Active ingredient(s) may be combined with matrix material prior to formation of the dosage form (e.g., a tablet).
  • active ingredient(s) may be coated on the surface of a particle, granule, sphere, microsphere, bead or pellet that comprises the matrix material. Such coating may be achieved by conventional means, such as by dissolving the active ingredient(s) in water or other suitable solvent and spraying.
  • additional ingredients are added prior to coating (e.g., to assist binding of the active ingredient(s) to the matrix material or to color the solution).
  • the matrix may then be coated with a barrier agent prior to application of controlled-releasc coating. Multiple coated matrix units may, if desired, be encapsulated to generate the final dosage form.
  • a controlled release is achieved through the use of a controlled release coating (i.e., a coating that permits release of active ingredient(s) at a controlled rate in aqueous medium).
  • a controlled release coating i.e., a coating that permits release of active ingredient(s) at a controlled rate in aqueous medium.
  • the controlled release coating should be a strong, continuous film that is smooth, capable of supporting pigments and other additives, non-toxic, inert and tack-free.
  • Coatings that regulate release of the modulator include pH-independent coatings, pH-dependent coatings (which may be used to release modulator in the stomach) and enteric coatings (which allow the formulation to pass intact through the stomach and into the small intestine, where the coating dissolves and the contents are absorbed by the body), it will be apparent that multiple coatings may be employed (e.g., to allow release of a portion of the dose in the stomach and a portion further along the gastrointestinal tract). For example, a portion of active ingredient(s) may be coated over an enteric coating, and thereby released in the stomach, while the remainder of active ingredaria(s) in the matrix core is protected by the enteric coating and released further down the GI tract.
  • pH dependent coatings include, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthaiate, hydroxypropylmelhylcellulose phthalate, methacryl ⁇ c acid ester copolymers and zein.
  • the coating is a hydrophobic material, preferably used in an amount effective to slow the hydration of the gelling agent following administration.
  • Suitable hydrophobic materials include alkyl celluloses (e.g., ethylcellulose or carboxymethylcellulose), cellulose ethers, cellulose esters, acrylic polymers (e.g., poly(acrylic acid), poly(methacrylic acid), acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxy ethyl methacrylates. cyanoelhyl methacrylate, methacrylic acid alkamide copolymer, po!y(methyl methacrylate), polyacry!
  • amide ammonio methacrylate copolymers, aminoalkyi methacrylate copolymer, poly(methacrylic acid anhydride) and glycidyl methacrylate copolymers) and mixtures of the foregoing.
  • Representative aqueous dispersions of ethylcellulose include, for example, AQUACOATCg ) (FMC Corp., Philadelphia, PA) and SURELEASE® (Coiorcon, Inc.. West Point, PA), both of which can be applied to the substrate according to the manufacturer's instructions.
  • Representative acrylic polymers include, for example, the various EUDRAG1T® (Rohm America, Pjscatavvay, NJ) polymers, which may be used singly or in combination depending on the desired release profile, according to the manufacturer's instructions.
  • the physical properties of coatings that comprise an aqueous dispersion of a hydrophobic material may be improved by the addition or one or more plasticizers.
  • Suitable plasticizers for alkyl celluloses include, for example, dibutyl sebacate, diethyl phthalate, trielhyl citrate, tributyl citrate and triacetin.
  • Suitable plasticizers for acrylic polymers include, for example, citric acid esters such as triethyl citrate and tributyl citrate, dibutyl phthalate, polyethylene glycols, propylene glycol, diethyl phthalate, castor oil and triacetin.
  • citric acid esters such as triethyl citrate and tributyl citrate, dibutyl phthalate, polyethylene glycols, propylene glycol, diethyl phthalate, castor oil and triacetin.
  • Controlled-release coatings are generally applied using conventional techniques, such as by spraying in the form of an aqueous dispersion.
  • the c ⁇ ating may cuiiipnse poics Oi uidu ⁇ eli to facilitate release of active ingredient.
  • Pores and channels may be generated by well known methods, including the addition of organic or inorganic material that is dissolved, extracted or leached from the coating in the environment of use.
  • Certain such pore-forming materials include hydrophilic polymers, such as hydroxyalkylcelluloses (e g., hydroxypropylmethylcellulose).
  • a controlled release coating may include one or more orifices, which may be formed my methods such as those described in US Patent Nos. 3.845,770: 4,034,758; 4,077,407; 4,088,864; 4.783,337 and 5,071.607. Controlled-release may also be achieved through the use of transdermal patches, using conventional technology (see, e.g , US Patent No. 4,668,232).
  • controlled release formulations may be found, for example, in US Patent Nos. 4,572,833; 4,587, 1 17; 4,606,909; 4,610,870; 4,684,516; 4,777,049: 4,994,276; 4,996,058; 5, 128,143; 5,202,128; 5,376,384; 5,384,133; 5,445,829; 5,510, 1 19; 5,618,560; 5,643.604; 5,891 ,474; 5,958,456; 6,039,980; 6, 143,353; 6,126,969; 6,156,342; 6,197,347; 6,387,394; 6,399,096; 6,437,000; 6,447,796; 6,475,493; 6,491,950; 6,524,615; 6,838,094; 6,905.709; 6,923,984; 6,923,988; and 6,91 ] , 217; each of which is hereby incorporated by reference for its teaching of the
  • a compound provided herein may be conveniently added to food or drinking water (e.g., for administration to non-human animals including companion animals (such as dogs and cats) and livestock).
  • Animal feed and drinking water compositions may be formulated so that the animal takes in an appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to feed or drinking water.
  • Benzimidazole carboxamide derivatives provided herein are generally administered in a therapeutically effective amount.
  • Preferred systemic doses are no higher than 50 mg per kilogram of body weight per day (e.g., ranging from about 0.001 mg to about 50 mg per kilogram of body weight per day), with oral doses generally being about 5-20 fold higher than intravenous doses (e.g., ranging from 0.01 to 40 mg per kilogram of body weight per day).
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage unit will vary depending, for example, upon the patient being treated and the particular mode of administration. Dosage units will generaily contain from about 10 ⁇ g to about 500 mg of an active ingredient. Optimal dosages may be established using routine testing, and procedures that are well known in the art.
  • compositions provided herein may, but need not, further comprise one or more additional pharmaceutical agents, such as an anti-inflammatory agent or analgesic.
  • Anti- inflammatory agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), nonspecific and cyclooxygenase-2 fCOX-2) specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, Icilunornide, cyclosporins A, IM gold, minocycline, azathioprine, tumor necrosis factor (TNF) receptor antagonists, soluble TNF alpha receptor (etanercept), anti ⁇ TNF alpha antibodies (e.g., infliximab and ada ⁇ mumab), anti-C5 antibodies, interleukin-1 (IL-I) receptor antagonists (e.g., anakmra or IL-I trap), IL-18 binding protein, CTLA4-Ig (e.g., abatacept),
  • anti-IL- 15 monoclonal antibody CDP 484, CDP 870, chemokine receptor antagonists, selective iNOS inhibitors. p38 kinase inhibitors, integrin antagonists, angiogenesis inhibitors, and TMI-I dual inhibitors.
  • Further anti-inflammatory agents include meloxicam, rofecoxib, celecoxib, etoricoxib. parecoxib. vaidecoxib and tilicoxib.
  • NSAIDs include, but are not limited to, ibuprofen. flurbiprofen, naproxen or naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin.
  • NSAIDs consists of compounds that inhibit cyclooxygenase (COX) enzymes: such compounds include celecoxib and rofecoxib. NSAIDs further include salicylates such as acetylsalicylic acid or aspirin, sodium salicylate, choline and magnesium salicylates, and salsalate, as well as corticosteroids such as cortisone, dexamethasone.
  • COX cyclooxygenase
  • NSAIDs further include salicylates such as acetylsalicylic acid or aspirin, sodium salicylate, choline and magnesium salicylates, and salsalate, as well as corticosteroids such as cortisone, dexamethasone.
  • methylprednisolone, prednisolone, prednisolone sodium phosphate, and prednisone are also antiinflammatory agents, and are listed above.
  • Other such medications include narcotic agents which typically act at one or more opioid receptor subtypes (e.g , ⁇ , K and/or ⁇ ), preferably as agonists or partiai agonists.
  • opioid receptor subtypes e.g , ⁇ , K and/or ⁇
  • Such agents include opiates, opiate derivatives and opioids, as well as pharmaceutically acceptable salts and hydrates thereof.
  • narcotic analgesics include, within preferred embodiments, alfentanil, alphaprodme, anileridine, bezitramide, buprenorphine, butorphanol, codeine, diacetyldihydromorphine, diacetylmorphine, dihydrocodeine, diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone, hydromorphone, isomethadone, levomethorphan, levorphane, levorphanol, meperidine, metazocine, methadone, methorphan, metopon, morphine, nalbuphine, opium extracts, opium fluid extracts, powdered opium, granulated opium, raw opium, tincture of opium, oxycodone, oxymorphone, paregoric, pentazocine, pethidine, phenazocine, piminodine, propoxyphene, racemethorphan
  • narcotic analgesic agents include acetorphine, acetyldihydrocodeme. acetyl methadol, ailylprodine, alphracetylmethadol, alphameprodine, alphamethadol, benzethidme, benzylmorphine, betacetylmethadol, betameprodme. betamethadol, betaprodinc, clonitazene, codeine methylbromide. codeine-N-oxide. cyprenorphine, desomorphine, dextromoramide. diampromide.
  • dihydiornoiphine uirneaO ⁇ adol, diniepheptanol, dimctiiyltliitunubutciic. dioxaphetyl butyrate, dipipanone, drotebanol, ethanol, cthylmethylthiambutene. etonitazene, etorphine. etoxeridine, furethidine. hydromorphinol, hydrox ⁇ pethidine, ketobcmidone, levomoramide. ievophe ⁇ acylraorphan. methyldesorphine. methyldihydromorphine, morpheridine.
  • morphine methylpromide morphine methylsulfonate
  • morphine-N-oxide mjrophin
  • naloxone naityhexone
  • nicocodeine nicomorphine. noracymethadol. norlevorphanol. normethadone, normorphine, ⁇ orpipanone, pentazocaine, phenadoxone. phenampromide, phenomorphan. phenoperidine. piritramide. pholcodine. proheptazoine. properidine. propiran, racemoramide, thebacon. trimeperidine and the pharmaceutically acceptable salts and hydrates thereof.
  • analgesic agents include, for example acetaminophen (paracetamol): aspirin and other NSA ⁇ Ds described above; NR2B antagonists; capsaicin receptor antagonists: anti-migraine agents; anticonvulsants such as oxcarbazepine and carbarn azepine; antidepressants (such as TCAs, SSRIs.
  • analgesic agents include CB2-receptor agonists, such as AM1241. capsaicin receptor antagonists and compounds that bind to the ⁇ 2 ⁇ subunit of voltage-gated calcium channels, such as gabapentin and pregabalin.
  • Representative anti-migraine agents for use in combination with a B; modulator provided herein include CQRP antagonists, ergotamines and 5-HTj agonists, such as sumatripan, naratriptan, zolmatriptan and rizatriptan.
  • compositions may be packaged for treating conditions responsive to B] modulation (e.g., treatment of pain, inflammation or other disorder(s) recited herein).
  • Packaged pharmaceutical preparations generally comprise a container holding a therapeutically effective amount of a pharmaceutical composition as described above and instructions (e.g., labeling) indicating that the composition is to be used for treating a condition responsive to B] modulation in a patient (e.g., pain or other disorder as indicated herein).
  • a packaged pharmaceutical preparation comprises one or more benzimidazole carboxamide derivatives provided herein and one or more additional agents in the same package, either in separate containers within the package or in the same container (i.e., as a mixture).
  • Preferred mixtures are formulated for oral administration (e.g., as pills, capsules, tablets or the like).
  • the package comprises a label bearing indicia indicating that the components are to be taken together for the treatment of pain.
  • the present invention provides methods for treating a condition responsive to B 1 modulation in a patient.
  • the patient may be afflicted with such a condition, or may be free of symptoms but considered at risk for developing such a condition.
  • a condition is ''responsive to Bj modulation 11 if the condition or symptom(s) thereof are alleviated, attenuated, delayed or otherwise improved by modulation of Bi activity.
  • such methods comprise administering to the patient a therapeutically effective amount of at least one benzimidazole carboxamide derivative as provided herein.
  • Conditions responsive to Bi modulation include, for example pain; inflammation including neuroinflammation (such as atherosclerosis), inflammation associated with airway diseases (e.g., asthma, including allergic asthma, exercise-induced bronchoconstriction, occupational asthma, and other non-allergic asthmas), and inflammatory skin disorders (e.g., psoriasis and eczema)); respiratory disorders including bronchoconstriction.
  • asthma chronic obstructive pulmonary disease (e.g., emphysema), chronic cough (including ACE-inhibitor cough), adult respiratory distress syndrome, bronchitis, pneumonia, allergic rhinitis and vasomotor rhinitis; vascular edema (including diabetes- related vascular disease); and epilepsy.
  • Other conditions responsive to Bi modulation include diabetes (e.g., type II or non insulin dependent, as well as diabetic vasculopathy, diabetic neuropathy, diabetic retinopathy, post capillary resistance and symptoms associated with insuiitis), seizure disorders (e.g., epilepsy), multiple sclerosis, liver disease, cardiovascular disorders (e.g., atherosclerosis, congestive heart failure and myocardial infarction), neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease) rheumatoid arthritis, infection, cancer, cranial trauma, rhinitis, septic shock, endotoxic and pancreatic shock, anaphylaxis, inflammatory bowel disease, irritable bowel syndrome, pancreatitis, cystitis, uveitis, vascular permeability, gingivitis, osteoporosis, benign prostatic hyperplasia, hyperactive bladder, cerebral edema, vasodilation, hypotension associated with sepsis
  • Pain that may be treated using the B 1 modulators provided herein may be chronic or acute and includes, but is not limited to, peripheral nerve-mediated pain (especially neuropathic pain, such as pain due to diabetes, postherpetic neuralgia, nerve injury, vulvodynia, root avulsions, painful traumatic neuropathy and painful polyneuropathy).
  • peripheral nerve-mediated pain especially neuropathic pain, such as pain due to diabetes, postherpetic neuralgia, nerve injury, vulvodynia, root avulsions, painful traumatic neuropathy and painful polyneuropathy.
  • Compounds provided herein may also be used in the treatment of, for example, viscera!
  • pain e.g., pancreatitis, interstitial cystitis and renal colic
  • persistent hyperalgesia inflammatory pain, repetitive motion pain, carpel tunnel syndrome, perioperative pain, algesia, oral neuropathic pain, toothache (dental pain), denture pain, ocular pain, postherpetic neuralgia, diabetic neuropathy, chemotherapy-induced neuropathy, reflex sympathetic dystrophy, trigeminal neuralgia, bone and joint pain (e.g., pain associated with osteoarthritis), rheumatoid arthritis, myofascial pain (e.g., muscular injury and fibromyalgia), Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome and/or pain associated with nerve and root damage, including as pain associated with peripheral nerve disorders (e.g., nerve entrapment and brachial plexus avulsions, amputation, peripheral neuropathies including bilateral peripheral neuropathy, tic douloureux, atypical facial
  • Additional neuropathic pain conditions include causalgia (reflex sympathetic dystrophy - RSD, secondary to injury of a peripheral nerve), neuritis (including, for example, sciatic neuritis, peripheral neuritis, polyneuritis, optic neuritis, postfebrile neuritis, migrating neuritis, segmental neuritis and Gombault's neuritis), neuronitis, neuralgias (e.g., those mentioned above, cervicobrachial neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngial neuralgia, migranous neuralgia, idiopathic neuralgia, intercostals neuralgia, mammary neuralgia, mandibular joint neuralgia, Morton's neuralgia, nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia, splenopalatine neuralg
  • Headache including headaches involving peripheral nerve activity may also be treated as described herein.
  • Such headache pain includes, for example, sinus, cluster (i.e., migranous neuralgia) and tension headaches, migraine, temporomandibular pain and maxillary sinus pain.
  • migraine headaches may be prevented by administration of a compound provided herein as soon as a pre-migrainous aura is experienced by the patient.
  • Charcot's pains intestinal gas pains, ear pain, heart pain, muscle pain, eye pain, orofacial pain (e.g., odontalgia), abdominal pain, gynaecological pain (e.g., menstrual pain, dysmenorrhoea, pain associated with cystitis, labor pain, chronic pe ⁇ vic pain, chronic prostitis and endometriosis), acute and chronic back pain (e.g., lower back pain), gout, scar pain, hemorrhoidal pain, dyspeptic pains, pain associated with angina, nerve root pain, "non-painful" neuropathies, complex regional pain syndrome, homotopic pain and heterotopic pain — including pain associated with carcinoma, often referred to as cancer pain (e.g., in patients with bone cancer), pain (and inflammation) associated with venom exposure (e.g., due to snake bite, spider bite, or insect sting) and trauma associated pain (e.g., post-surgical pain
  • cancer pain e.g., in
  • Additional pain conditions that may be treated as described herein include pain associated with respiratory disorders as described above, autoimmune diseases, immunodeficiency disorders, hot flashes, inflammatory bowel disease, gastroesophageal reflux disease (GERD), irritable bowel syndrome and/or inflammatory bowel disease.
  • pain treated with B 1 modulators provided herein is inflammatory pain, acute pain, dental pain, back pain, surgical pain, headache, neuropathic pain or pain from osteoarthritis or trauma.
  • compounds provided herein may be administered alone or in combination with one or more additional agents that are suitable for treating the disorder of interest.
  • the compound(s) and additional agent(s) may be present in the same pharmaceutical composition, or may be aum ⁇ iisteied sepaiateiy in eithes ordei.
  • Repiese ⁇ tatrve anti-inflammatory agents and analgesics for use in combination therapy include those indicated above.
  • B; modulators provided herein may be used within combination therapy for the treatment of conditions involving pain and/or inflammatory components.
  • Such conditions include, for example, autoimmune disorders and pathologic autoimmune responses known to have an inflammatory component including, but not limited to, arthritis (especially rheumatoid arthritis), psoriasis, Crohn's disease, lupus erythematosus, irritable bowel syndrome, tissue graft rejection, and hyperacute rejection of transplanted organs.
  • Other such conditions include trauma (e.g., injury to the head or spinal cord), cardio- and cerebrovascular disease and certain infectious diseases.
  • a Bi modulator is administered to a patient along with an additional analgesic and/or anti-inflammatory agent.
  • the Bj modulator and additional analgesic and/or anti-inflammatory agent may be present in the same pharmaceutical composition, or may be administered separately in either order.
  • Administration to the patient can be by way of any means discussed above, including oral, topical, nasal or transdermal administration, or intravenous, intramuscular, subcutaneous, intrathecal, epidural, intracerebrove ⁇ t ⁇ ' cular or like injection.
  • Oral administration is preferred in certain embodiments (e.g., formulated as pills, capsules, tablets or the like).
  • Treatment regimens may vary depending on the compound used and the particular condition to be treated. In general, a dosage regimen of 4 times daily or less is preferred, with 1 or 2 times daily particularly preferred. It will be understood, however, that the specific dose and treatment regimen 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 and diet of the patient the time of administration, the route of administration, the rate of excretion, any drug combination and the severity of the particular disease undergoing therapy. Dosages are generally as described above: in general, the use of the minimum dose sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using medical or veterinary criteria suitable for the condition being treated or prevented.
  • Suitable dosages for Bj modulators are generally as described above. Dosages and methods of administration of any additional agent(s) (e.g., anti-inflammatory and/or analgesic agents) can be found, for example, in the manufacturer's instructions or in the Physician's Desk Reference. In certain embodiments, combination administration results in a reduction of the dosage of the additional agent required to produce a therapeutic effect (i.e., a decrease in the minimum therapeutically effective amount). Thus, preferably- the dosage of additional agent in a combination or combination treatment method of the invention is less than the maximum dose advised by the manufacturer for administration of the agent without combination with a compound of Formula 1.
  • any additional agent(s) e.g., anti-inflammatory and/or analgesic agents
  • combination administration results in a reduction of the dosage of the additional agent required to produce a therapeutic effect (i.e., a decrease in the minimum therapeutically effective amount).
  • the dosage of additional agent in a combination or combination treatment method of the invention is less than the maximum dose advised by the manufacturer for administration of the
  • this dose is less than 3 A, even more preferably less than ' ⁇ , and highly preferably less than ] A of the maximum dose, while most prefeiabh the dose is less than 10% ⁇ f the maximum uuse advised by the manufacturer for administration of the agcnt(s) when administered without combination administration as described herein. It will be apparent that the dose of compound as provided herein needed to achieve the desired effect may similarly be affected by the dose and potency of the additional agent.
  • the present invention provides a variety of non-pharmaceutical in vitro and in vivo uses for the compounds provided herein.
  • such compounds may be labeled and used as probes for the detection and localization of Bi (in samples such as cell preparations or tissue sections, preparations or fractions thereof).
  • compounds provided herein that comprise a suitable reactive group such as an aryl carbonyl. nitro or azide group
  • compounds provided herein may be used as positive controls in assays for receptor activity, as standards for determining the ability of a candidate agent to bind to B!
  • a compound may be labeled using any of a variety of well known techniques (e.g., radiolabeled with a radionuclide such as tritium, as described herein), and incubated with a sample for a suitable incubation time (e.g., determined by first assaying a time course of binding).
  • a radionuclide such as tritium, as described herein
  • unbound compound is removed (e.g., by washing), and bound compound detected using any method suitable for the label employed (e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups).
  • any method suitable for the label employed e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups.
  • a matched sample containing labeled compound and a greater (e.g., 10-fold greater) amount of unlabeled compound may be processed in the same manner. A greater amount of detectable label remaining in the test sample than in the control indicates the presence of B] in the sample.
  • Detection assays including receptor autoradiography (receptor mapping) of B 1 in cultured cells or tissue samples may be performed as described by Kuhar in sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998) John Wiley & Sons, New York.
  • Mass spectroscopy data in the following Examples is Electrospray MS, obtained in positive ion mode using a Micromass Time-of-Flight LCT (Micromass, Beverly MA), equipped with a Waters 600 pump (Waters Corp.; Milford, MA), Waters 996 photodiode array detector, and a Gilson 215 autosampler (Gilson, Inc.; Middleton, WI). MassLynx (Advanced Chemistry Development. Inc; Toronto, Canada) version 4.0 software with Open Lynx Global ServerTM, OpenLynxTM and AutoLynxTM processing is used for data collection and analysis.
  • Sample volume of 1 microliter is injected onto a 50x4.6mm Chromolith SpeedROD RP-18e column (Merck KGaA, Darmstadt, Germany), and eluted using a 2-phase linear gradient at a flow rate of 6 mf/min. Sample is detected using total absorbance count over the 220-340nm UV range.
  • This Example illustrates the preparation of Bptransfected cells for use in Bi binding and modulation assays (Examples 6 and 7).
  • RNA is isolated as described by Chomzynski et al. ( 1987) Anal. Bi ⁇ chem. /62: 156-159.
  • a cDNA encoding Bi is cloned from the total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with the following oligonucleotides:
  • Primer 1 GGCGCT AGCCACC ATGGCATCCTGGCCCCCTC (SEQ ID NO: 1)
  • Primer 2 AGCCGTCCCAGATCTGAAC (SEQ ID NO:2)
  • Primer 4 CGGAGCTCTTAATTCCGCCAGAAAAGTTGGA (SEQ ID NO:4) Primer pairs 1 & 2 and 3 & 4 ace used to generate overlapping cDNA fragments corresponding to the entire protein coding sequence of cynomoigus macaque B 1 cDNA are isolated and linked to form the fuli-Iength coding sequence (GenBank Accession Number AY788905).
  • the construct is cloned into pcDNA 3.1 (Invitrogen. Carlsbad, CA) and transfecled into Chinese hamster ovary (CHO) cells using Lipofectam ⁇ ne (Invitrogen), resulting in cynomoigus macaque B 1 -expressing CHO cells.
  • the construct is cloned into pBAKPAK9 (Clontech, Mountain View, CA) and transfected into Sf9 cells to generate clonal baculovirus stocks.
  • Clonal cell lines stably expressing the cynomoigus macaque B 1 receptor are selected in G4I 8.
  • a single clonal line that exhibits high levels of receptor expression is chosen for use in binding and calcium mobilization assays (Examples 6 and 7).
  • Clonal baculovirus stocks are used to infect Sf9 cells such that the infect d cells express high levels of recombinant B] receptors. These cells are used in radioligand binding assays (Example 6).
  • This Example illustrates a representative Bi receptor binding assay that may be used to determine the binding affinity of compounds for B 1 .
  • ⁇ MR-90 cells which endogenously express human B 5 , are seeded into 24 well plates at
  • CHO cells stably expressing rat B 1 are seeded into 24 well plates at 200,000 cells per well and cultured overnight. The ceils are then washed 3 times with phosphate buffered saline (PBS). One hundred fifty microliters of binding buffer (50 mM Tris 7.4, 0.14 mg/mL, bacitracin, and 1 mg/mL BSA) is added to each well.
  • PBS phosphate buffered saline
  • test compound Various concentrations of test compound are added to each well from DMSO solutions such the final DMSO concentration is 1% by volume; some wells receive DMSO only, and some wells receiveDMSO plus 10 ⁇ MdesArg 10 Kallidin to define non-specific binding. All wells then receive 0.3nM
  • SiP cells infected with a baculovirus carrying the coding sequence for cynomolgus macaque Bi are harvested by centrilugation and frozen at -80 0 C. Pellets are subsequently resuspended on ice in Tn s buffered saline (TBS: 50 mM Tris (pH 7.4). 120 mM NaCl). and cells are homogenized using a polytron for 30 seconds. The crude membrane fraction is collected by centrifugation at 20,000 ipm. Membranes are washed two times with TBS and collected by centrifugation each time.
  • Protein content of the membranes is determined after the last wash and the concentration is adjusted to 0.7 ⁇ g/uL with binding buffer (50 mM Tris 7,4, 0.14 mg/mL bacitracin, and 1.0 mg/niL BSA).
  • binding buffer 50 mM Tris 7,4, 0.14 mg/mL bacitracin, and 1.0 mg/niL BSA.
  • Some wells receive DMSO only, and some welis receive DMSO plus 10 ⁇ M desArg 10 Kallidin to define non-specific binding.
  • the 96 well plates are allowed to sit for 2 h at room temperature.
  • Membrane proteins are then harvested by filtration onto GF/C f ⁇ ltermats (PerkinElmer) pre-soaked for 1 hr in 0.5 % polyethylenimine. After filtration, filters are dried and then counted in a Beta plate counter. The number of counts present in the scintillation fluid is plotted as a function of antagonist compound concentration and fitted to a logistical equation using SigmaPIot (Systat Software, Point Richmond, CA) to determine each compound's IC 5 0 and K 1 (e.g., as described by Szallasi, el al (1993) J. Pharmacol. Exp. Ther. 266:678-83).
  • This Example illustrates representative calcium mobilization assays for use in evaluating test compounds for agonist and antagonist activity.
  • Cynomolgus macaque Bj -express ing CHO cells (Example 5) are plated in a 96 well plate. The cells are cultured for 1 day, after which culture media is emptied from the plate and replaced with 50 ⁇ l of KRH (Krebs-Ringer HEPES buffer: 25 mM HEPES, 5 mM KCl, 0.96 mM NaH 2 PO 4 , 1 mM MgSO 4 , 2 mM CaCl 3 . 5 mM glucose. 1 mM probenecid. pH 7.4) supplemented with the calcium- sensitive fluorescent dye Fluo4-AM (5 ⁇ g/mi: Teflabs, Austin, TX).
  • KRH Kerat-Ringer HEPES buffer: 25 mM HEPES, 5 mM KCl, 0.96 mM NaH 2 PO 4 , 1 mM MgSO 4 , 2 mM CaCl 3 . 5 mM glucose. 1 mM probene
  • the cells are then incubated at 37 0 C in an environment containing 5% CO 2 . After the 1 hour incubation, the dye solution is removed from the plate, the plate is washed once with KRH, and ] 00 ⁇ L KRH is added. DEI ERMINAT ION OF B 1 AGONIST EC 50
  • this assay is also used to assess B
  • test compounds are added to the cell plate prepared as described above in 100 ⁇ L KRH + 2% DMSO, such that the final volume in each well is 200 microliters and the final DMSO concentration is 1%.
  • the EC 50 concentration of desArg ! °Kallidin is then added to each well of plates containing test compound to determine the extent to which each test compound inhibits an agonist-induced B 1 response.
  • the maximum fluorescent response is plotted as a function of test compound concentration in order to determine the IC5 0 (concentration required to inhibit 50% of the effect of agonist) for each compound at B 1 .
  • Antagonists of Bj decrease this response by at least about 20%, preferably by at least about 50%.
  • the data is analyzed as follows. First, the average maximum relative fluorescent unit (RFU) response from negative control wells (no agonist) is subtracted from the maximum response detected for each of the other experimental wells. Second, average maximum RFU response is calculated for the positive control wells (agonist wells).
  • RFU relative fluorescent unit
  • % inhibition data is plotted as a function of test compound concentration and test compound IC 1 o is determined using a linear regression in which x is ln(concenlration of test compound) and y is ln(percent inhibition/(100 - percent inhibition). Data with a percent inhibition that is greater than 90% or less than 15% are rejected and are not used in the regression.
  • the IC 50 is
  • This Example illustrates the evaluation of compound toxicity using a Madin Darby canine kidney (MDCK) cell cytotoxicity assay.
  • test compound 1 ⁇ L is added to each well of a clear bottom 96-well plate (Packard, Meriden, CT) to give final concentration of compound in the assay of 10 ⁇ M, 100 ⁇ M or 200 ⁇ M. Solvent without test compound is added to control wells.
  • MDCK cells ATCC no. CCL-34 (American Type Culture Collection, Manassas, VA), are maintained in sterile conditions following the instructions in the ATCC production information sheet. Confluent MDCK ceils are trypsinized, harvested, and diluted to a concentration of 0.1 x 10 6 ceils/mL with warm (37 0 C) medium (VITACELL Minimum Essential Medium Eagle, ATCC catalog # 30-
  • the ATP-LITE-M Luminescent ATP detection kit is generally used according to the manufacturer's instructions to measure ATP production in treated and untreated MDCK cells. PACKARD ATP LITE-M reagents are allowed to equilibrate to room temperature. Once equilibrated, the lyophilized substrate solution is reconstituted in 5.5 mL of substrate buffer solution (from kit). Lyophilized ATP standard solution is reconstituted in deionized water to give a 10 mM stock.
  • PACKARD substrate solution 50 ⁇ L is added to all wells, which are then covered, and the plates are shaken at approximately 700 rpm on a suitable shaker for 2 min.
  • a white PACKARD sticker is attached to the bottom of each plate and samples are dark adapted by wrapping plates in foil and placing in the dark for 10 min.
  • Luminescence is then measured at 22°C using a luminescence counter (e.g., PACKARD TOPCOLTNT Microplate Scintillation and Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levels calculated from the standard curve.
  • ATP levels in cells treated with test compound(s) are compared to the levels determined for untreated cells.
  • Cells treated with 10 ⁇ M of a preferred test compound exhibit ATP levels that are at least 80%, preferably at least 90%, of the untreated cells.
  • a 100 ⁇ M concentration of the test compound is used, cells treated with preferred test compounds exhibit ATP levels that are at least 50%, preferably at least 80%). of the ATP levels detected in untreated cells.

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Abstract

Benzimidazole carboxamide derivatives of Formula I are provided: Formula (I) in which the variables are as described herein. Such compounds may be used to modulate bradykinin receptor activity in vivo or in vitro, and are particularly useful in the treatment of conditions responsive to B1 modulation in humans, domesticated companion animals and livestock animals, including inflammation and pain. Pharmaceutical compositions and methods for using them to treat such disorders are provided, as are methods for using such ligands for receptor localization studies and various in vitro assays.

Description

BENZIMIDAZOLE CARBOXAMIDE DERIVATIVES
FIELD OF THE INVENTION
This invention relates generally to benzimidazole carboxamide derivatives, and to the use of such compounds to treat conditions responsive to bradykinin receptor-1 (B]) modulation. The invention further relates to the use of such compounds as reagents for the identification of other agents that bind to Bi, and as probes for the detection and localization of B].
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application 60/825,308, filed September 12, 2006, which is hereby incorporated by reference in its entirety.
DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 B1 PCR primer number 1
SEQ ID NO:2 B1 PCR primer number 2
SEQ ID NO:3 B1 PCR primer number 3 SEQ ID NO:4 B1 PCR primer number 4
BACKGROUND OF THE INVENTION
Millions of people throughout the world suffer debilitating and incapacitating pain. Current treatments for pain can be beneficial, but such treatments are not completely effective, and typically have undesirable side effects. For example, non-steroidal anti-inflammatory drugs are commonly used, but are only moderately effective against pain, and have serious renal and gastrointestinal side effects at high doses. Opiates, such as morphine, are potent analgesics, but their usefulness is limited because of adverse side effects, such as physical addictiveness and withdrawal properties, as well as respiratory depression, mood changes, and decreased intestinal motility with concomitant constipation, nausea, vomiting, and alterations in the endocrine and autonomic nervous systems. There is thus a need for effective agents for the treatment of pain.
Bradykinin (BK) is a πonapeptide (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) that functions in cardiovascular homeostasis, contraction and relaxation of smooth muscles, inflammation and pain. The effects of BK are mediated by specific G protein-coupled BK receptors, of which there are at least two distinct subtypes termed Bi and B2. The B2 receptor is expressed constitutively in a variety of tissues. In contrast, the Bi receptor is inducibly expressed in response to pathophysiological conditions such as inflammation, pain, trauma, bacterial infection, burns and shock.
Accordingly, Bj is a particularly attractive drug target for these and other conditions, and agents that act at this receptor may be targeted specifically to injured tissues, with minimal effects in normal tissues. In addition, compounds that bind to B1 and/or modulate the activity of Bj also find use as research tools.
There is thus a need in the art for small molecule modulators of B1 activity. The present invention fulfills this need and provides further related advantages.
SUMMARY OF THE INVENTION
The present invention provides benzimidazole carboxamide derivatives that satisfy Formula I:
Figure imgf000003_0001
or are a pharmaceutically acceptable salt, solvate (e.g., hydrate) or ester of such a compound. Within Formula I:
A, B, D and E are each CH or N; preferably such that no more than one of A, B, D and E is N; Y is a group of the formula (CH2)I-Q-(CH2)P, which is optionally substituted and is preferably substituted with from 0 to 4 substituents independently chosen from (i) amino, hydroxy, cyano, C]-C6alkyl, C2-C6aikenyl, C3-Cc,alkynyl and C1-Cβhaloalkyl; and (ii) substituents of the same carbon atom or adjacent carbon atoms that are taken together to form C3-C6cycioalkyl, wherein:
O is absent, CHR10, CH=CH, O, S or NR10; wherein Ri0 is hydrogen, CpQaikyl or taken together with R3 to form a 4- to 7-membered optionally substituted heterocycloaikyl; and r and p are independently chosen integers ranging from 0 to 6; preferably such that the sum of r and p ranges from 1 to 6;
R1 represents from 0 to 4 substituents; preferably each substituent is independently chosen from halogen, hydroxy, cyano, amino, nitro, aminocarbonyl, aminosulfonyl, -COOH, C1-C6a!kyl, C2- Qalkenyl, C2-C6alkynyl, (C3-C3cyc!oa!kyl)Co-C4a!kyl, C1-C6alkoxy; C1-C6alky!thio, C,-
Qsafkylsuifϊnyl, C1-Ceaikoxycarbonyl, C]-C6alkylsu]fonylCo-C4alkyI, and mono- or di-(C1-
C6alkyl)aminoCo-C4alkyl;
R2 is C1-C6alkyl, CrC6alkenyl, C2-C6a]kynyl, (C3-C8cycloalkyI)C0-C4alkyl, mono- or di-(C,-
C6aIkyl)aminoC1-C4alkyl5 (4- to 7-membered beterocycloalkyl)Co-C4aIkyl, phenyIC0-C4alkyl or (5- or 6-membered heteroaiyl)Co-C4aikyl, each of which is substituted with from 0 to 4 substituents independently chosen from amino, halogen, hydroxy, cyano, oxo, C1-Qalkyl, (C3-
Cgcycloalkyl)Co-C4alkyl and C1-Cβalkoxy;
R3 is hydrogen, CrQalkyl or taken together with Ri0 to form a 4- to 7-membered optionally substituted heterocycloaikyl; and R4 and R5 are: (i) independently chosen from C1-C6alkyl, C2-C6alkenyl, C2-Qalkynyl, (C3-C3cycIoalkyI)Co- C4alkyl, (4- to 7-rnembered heterocycloaIkyl)C0-C4aikyl, phenylC0-C4alkyl and (5- to 10- membered heteroaiyl)Co-C4aIkyl; or
(ii) taken together to form a 4- to 7-menibered heterocycloalky!; each of which (i) and (ii) is optionally substituted and is preferably substituted with from 0 to 6 substituents independently chosen from:
(a) halogen, hydroxy, cyano, amino, nitro, -COOH, aminocarbonyi and oxo; and
(b) CrQaikyl, C2-C6alkenyl, C2-C6alkynyi, C1-C6alkoxy, (C3-C3cycloalkyl)Co-C4alkyl, mono- or di-(C1-C<$alkyl)amiπo, (4- to 7-membered heterocycloaikyl)Co-C4alkyl, ρhenylCo-C4alkyl, (5- or 6-membered heteroaryl)Co-C4alkyl, and substituents that are taken together to form a fused or spiro 5- to 7-membered ring; each of which (b) is substituted with from 0 to 4 substituents independently chosen from:
(1) amino, halogen, hydroxy, cyano, -COOH, aminocarbonyi and oxo; and
(2) C1-Qaikyi, C1-C6alkoxy, C2-C6alkyl ether, mono- or di-(C1-C6alkyJ)aminoC0-C4aIkyl,
Figure imgf000004_0001
(4- to 7-membered heterocycloalkyl)Co-C4alkyl, phenylC0-C4alkyl and (5- or ό-mernbered heteiOary!)C0-C4alkyl, each of which is substituted with from 0 to 4 substituents independently chosen from haiogen, oxo and C1-C4alkyl.
Within certain aspects, benzimidazole carboxamide derivatives of Formula I, and other Formulas provided herein, are B1 modulators and exhibit a K1 of no greater than 5 micromolar, 2 micromolar, 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar or 10 nanomolar in a Bi binding assay and/or have an EC50 or IC50 value of no greater than 5 micromolar, 2 micromolar, 1 micromolar, 500 nanomolar, 100 nanomolar, 50 nanomolar or 10 nanomolar in an assay for determination of Bi agonist or antagonist activity. In certain embodiments, B] modulators provided herein are Bi antagonists; preferably such antagonists exhibit no detectable Bj agonist activity.
Within certain aspects, benzimidazole carboxamide derivatives of Formula 1 are labeled with a detectable marker (e.g., radiolabeled or fluorescein conjugated).
The present invention further provides, within other aspects, pharmaceutical compositions comprising at least one benzimidazole carboxamide derivative of Formula ϊ in combination with a physiologically acceptable carrier or excipient.
The present invention further provides, within other aspects, pharmaceutical compositions comprising at least one benzimidazole carboxamide derivative as described herein in combination with a physiologically acceptable carrier or excipient. Methods are further provided for inhibiting agonist-induced B1 activity. Within certain such aspects, the inhibition takes place in vitro. Such methods comprise contacting a B1 receptor with at least one Bj antagonist as described herein, under conditions and in an amount or concentration sufficient to detectably inhibit agonist-induced B] activity. Within other such aspects, the Bi receptor is in a patient. Such methods comprise contacting ceils expressing a B] receptor in a patient with at least one B] antagonist as described herein in an amount or concentration that would be sufficient to detectabiy inhibit agonist-induced Bj activity in cells expressing a cloned B, receptor in vitro. The present invention further provides methods for treating a condition responsive to Bj receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of at least one benzimidazole carboxamide derivative of Formula 1.
Within other aspects, methods are provided for treating pain in a patient, comprising administering to a patient suffering from (or at risk for) pain a therapeutically effective amount of at least one benzimidazole carboxamide derivative of Formula I. Pain conditions that may be treated include, but are not limited to, inflammatory pain, acute pain, dental pain, back pain, surgical pain, headache, neuropathic pain, and pain associated with osteoarthritis or trauma.
Within further aspects, the present invention provides methods for determining the presence or absence of Bi in a sample, comprising: (a) contacting a sample with a benzimidazole carboxamide derivative of Formula 1 under conditions that permit binding of the compound to Bj; and (b) detecting a signal indicative of a level of the compound bound to Bj.
In yet another aspect, the present invention provides methods of preparing the compounds disclosed herein, including the intermediates.
These and other aspects of the present invention will become apparent upon reference to the following detailed description.
DETAILED DESCRIPTION
As noted above, the present invention provides benzimidazole carboxamide derivatives, which may be used in vitro or in vivo in a variety of contexts, as described herein.
TERMINOLOGY 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. 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. If 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. Certain compounds are described herein using a general formula that includes variables {e.g., Y, R]). 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 foπnula is defined independently at each occurrence. The term "benzimidazole carboxamide derivative" encompasses any compound that satisfies Formula I. This term further includes pharmaceutically acceptable salts, solvates (e.g., hydrates) and esters of such compounds.
A "pharmaceutically acceptable salt" of a compound recited herein is an acid or base salt that is 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. Specific pharmaceutically acceptable anions for use in salt formation include, but are not limited to. acetate, 2-acetoxybenzoate, ascorbate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carbonate, chloride, citrate, dihydrochloride, diphosphate, edetate, estolate (ethylsuccinate), formate, fumarate, gluceptate, gluconate, glutamate, giycolate, glycollylarsanilate, hexyiresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, hydroxymaleate, hydroxynaphthoate, iodide, isethionate, lactate, iactobionate, malate, m abate, mandeiate, methylbromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phenylacetate, phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, succinate, sulfamate, sulfanilate, sulfate, sulfonates including besylate (bcnzenesulfonate), camsylate (camphorsulfonate), edisyfate (ethane- 1 ,2-disulfonate), esylate (ethanesulfonate), 2-hydroxyethylsulfonate, mesylate (methanesuifonale), triflate
(trifluoromethanesulfonate) and tosylate (p-toluenesulfonate), tannate, tartrate, teoclate and triethiodide. Similarly, pharmaceutically acceptable cations for use in salt formation include, but are not limited to ammonium, benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, and metais such as aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. 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, methanol, isopropanol or acetonitrile, is preferred. It will be apparent that each compound provided herein may, but need not. be formulated as a solvate (e.g., a hydrate) or non-covalcnt complex. In addition, the various crystal forms and polymorphs are within the scope of the present invention. Also provided herein are prodrugs of the compounds provided herein, A "prodrug" is a compound that may not fully satisfy the structural requirements of a formula provided herein, but is modified in vivo, following administration to a patient, to produce a compound within the scope of such formula. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, amine or suifhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to yield the parent compounds.
As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon. Alkyl groups include groups having from 1 to 8 carbon atoms (CpCgalkyl), from 1 to 6 carbon atoms (CpQalkyl) and from 1 to 4 carbon atoms (C1-C4alkyI), such as methyl, ethyl, propyl, isopropyl, n-butyϊ, sec-butyl, fer/-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl or 3-methyipentyI. "C0-C4alkyl" refers to a single covalent bond (C0) or an alkylene group having 1, 2, 3 or 4 carbon atoms; "C0-C2alky " refers to a single covalent bond or a methylene or ethylene group.
"Alkylene" refers to a divalent alkyl group, as defined above. C1-C4alkylene is an alkylene group having 1, 2. 3 or 4 carbon atoms. "Alkenyl" refers to straight or branched chain alkene groups, which comprise at least one unsaturated carbon-carbon double bond. Alkenyl groups include C2-C8alkenyl, C1-Cgalkenyl and C2- C4alkenyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively, such as ethenyl, ally! or isopropenyl. "Alkynyl" refers to straight or branched chain alkyne groups, which have one or more unsaturated carbon-carbon bonds, at least one of which is a triple bond. Alkynyl groups include C1-C8alkynyl, C2-C6alkynyl and C2-C4a!kynyl groups, which have from 2 to 8, 2 to 6 or 2 to 4 carbon atoms, respectively.
A "cycloalkyl" is a saturated or partially saturated cyclic group in which all ring members are carbon, such as cyclopropyi, cyclobutyl, cyclopentyl, cyclohexyl and partially saturated variants thereof. Certain cycloalkyl groups are C3-C8cycloalkyl, in which the ring contains from 3 to 8 ring members, all of which are carbon. A "( C3-C8cycloalkyl)C0-C4alkyl" is a C3-C8cycloalkyi group linked via a single covalent bond or a C1-C4alkylene group. By "alkoxy", as used herein, is meant an alkyl group attached via an oxygen bridge (i.e., -O- alkyl). Alkoxy groups include C1-C^alkoxy and C1-C4alkoxy groups, which have from 1 to 6 or from
I to 4 carbon atoms, respectively. Methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert- butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and
3-methyIρentoxy are representative alkoxy groups.
Similarly, "alkylthio" refers to an alkyl group as attached via a sulfur bridge (i.e., -S-alkyl).
Alkylthio groups include C1-C6alkylthio and C1-C4aikylthio groups, which have from 1 to 6 or from I to 4 carbon atoms, respectively. "Alkylsulfmyl" refers to groups of the formula -(SO)-alkyl, in which the sulfur atom is the point of attachment. Alkytsulfinyl groups include C1-C6alkylsulfmyl and C1-C4alkylsulfϊnyl groups, which have from I to 6 or from 1 to 4 carbon atoms, respectively. "Alkyisulfonyj" refers to groups of the formula -{SO2)-aIkyL in which the sulfur atom is the point of attachment. Alkylsulfonyl groups include C1-Cβalkylsulfonyl and C1-Qalkylsulfony! groups, which have from 1 to 6 or from 1 to 4 carbon atoms, respectively. The term "(C1-CealkyisulfonylJCo-
C4alkyl refers to a C1-C6alkylsulfonyl that is linked via a single covaleπt bond or a C1-C4alkylene group (i.e., -(C0-C4alkylHSO2MC1-C6alkyl)).
The term "alkoxycarbonyl" refers to an alkoxy group linked via a carbonyl (i.e., a group having the general structure -C(=O)-O-aIkyl). Alkoxycarbonyl groups include C]-Cg, Q-Cβ and Q- C4alkoxycarbonyl groups, which have from 1 to 8, 6 or 4 carbon atoms, respectively, in the alkyl portion of the group. "C1 alkoxycarbonyl" refers to -Cf=O)-O-CH-,. "Alkylamino" refers to a secondary or tertiary amine that has the general structure -NH-alkyl or -N(alkyi)(alkyl), wherein each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Such groups include, for example, mono- and di-(C1-Csalkyi)amino groups, in which each C1-Csalkyl may be the same or different, as well as mono- and di~(C1-Q,alkyl)amino groups and mono- and di-(C1-C4alkyl)amino groups. "Alkylaminoalkyl" refers to an alkylamino group linked via an alkylene moiety (Le , a group having the general structure -aikyiene-NH-alkyl or -alkylene-N(alkyl)(alkyl)) in which each alkyl is selected independently from alkyl, cycloalkyl and (cycloalkyl)alkyl groups. Alkylaminoalkyl groups include, for example, mono- and di-(C;-CsaIkyl)aminoCrC6alkyl, and mono- and di-(C1- C6aIkyI)aminoC1-C1alkyl. "Mono- or di-(C1-C6aIky])aminoCo-C4aIkyl" refers to a mono- or di-(Ct- C6alkyl)amino group linked via a single covalent bond or a C1-C4alkylene group. The following are representative alkylaminoalkyl groups:
Figure imgf000008_0001
It will be apparent that the definition of "alkyl" as used in the terms "alkylamino" and "alkylaminoalkyl" differs from the definition of "alkyl" used for all other alkyi-containing groups, in the inclusion of cycloalkyl and (cyclnalkyl)aϋcyl groups (e g , (CrC~cyrlnaIkyl)Co-C2a!kyl) The term "aminocarbonyl" refers to an amide group (i.e., ™C(=O)NH2). Similarly, "aminosulfonyl" refers Io a sulfonamide group (i. e , -SO2NH2). The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "oxo," as used herein refers to an oxygen substituent of a carbon atom that results in the formation of a carbonyl group (C=O). An oxo group that is a substituent of a nonaromatic carbon atom results in a conversion of -CH2- to -C(=O)-. An oxo group that is a substituent of an aromatic carbon atom results in a conversion of -CH- to -C(=O)- and ma> result in a loss of aromaticity.
A "haloalkyl" is an aikyl group that is substituted with 1 or more independently chosen halogens (e.g., "C1-C8haloalkyl" groups have from 1 to 8 carbon atoms: "C1-C6haioalkj l" groups have from i to 6 carbon atoms). Examples of haloaikyi groups include, but are not limited to, mono-, di- or tri-fluoromethyϊ; mono-, di- or tri-chlororaethyl; mono-, di-, tri-. tetra- or penta-fluoroethyl; mono-, di-, tri-, tetra- or penta-chloroethyl; and 1,2,2,2-tetrafluoro-l-trifluoromethyl-ethyI. Typical haloalkyl groups are trifluoromethy] and difluoromethyl.
A dash ("-") that is not between two letters or numbers is used to indicate a point of attachment for a substituent. For example, -C(=O)NH2 is attached through the carbon atom.
Phenyl groups linked via a single covalent bond or Q-dalkylene group are designated pheny!C0-C4alkyl (e.g., phenylC,-C2alkyI, which includes benzyl, l~ρhenyl-ethyl and 2-phenyl-ethyl). When substituted, it will be apparent that such groups may be substituted on the ring portion and/or on the alkylene portion of the group. A "heterocycle" (also referred to herein as a "heterocyclic group") has from 1 to 3 fused, pendant or spiro rings, at least one of which is a heterocyclic ring (i.e., one or more ring atoms is a heteroatom independently chosen from oxygen, sulfur and nitrogen, with the remaining ring atoms being carbon). Typically, a heterocyclic ring comprises 1, 2, 3 or 4 heteroatoms; within certain embodiments each heterocyclic ring has 1 or 2 heteroatoms per ring. Each heterocyclic ring generally contains from 3 to 8 ring members (rings having from 4 or 5 to 7 ring members are recited in certain embodiments) and heterocycles comprising fused, pendant or spiro rings typically contain from 9 to 14 ring members. Certain heterocycles comprise a sulfur atom as a ring member; in certain embodiments, the sulfur atom is oxidized to SO or SC^- Heterocycles may be optionally substituted with a variety of substituents, as indicated. Certain heterocycles are 4- to 10-membered and comprise one or two rings; in certain embodiments, such heterocycles are monocyclic (e.g., 4- to 8-membered, 5- to 8-membered, 4- to 7-membered, or 5- or 6-membered).
Certain heterocycles are heteroaryi groups (i.e., at least one heterocyclic ring within the group is aromatic), such as a 5- to 10-membered heteroaryi (which may be monocyclic or bicyclic) or a 5- or
6-membered heteroaryi (e.g., tbtenyl, imidazolyl, pyridyl or pyrimidyl). Other heterocycles are heterocycloalky] groups (i.e., do not comprise an aromatic heterocyclic ring). Certain heterocycies may be linked by a single covalent bond or via an alkylene group, as indicated, for example, by the term "(4- to 7-mεmberεd
Figure imgf000009_0001
"(4- to 7-membered heterocycloalkyϊ)C1-
C2alkyl," for example, refers to a 4- to 7-membered heterocycloalkyl that is linked via a methylene or ethylene linker. When substituted, it will be apparent that substituent(s) may be attached on the ring and/or the alkylene linker.
A "substituent," as used herein, refers to a molecular moiety that is covaiently 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 group discussed herein that is covaiently bonded to an atom (such as a carbon or nitrogen atom) that is a ring member. The term "substitution" refers to replacing a hydrogen atom in a molecular structure with a substituent as described above, such that the valence on the designated atom is not exceeded, and such that a chemically stable compound (i.e., a compound that can be isolated, characterized, and tested for biological activity) results from the substitution.
Groups that are "optionally substituted'1 are unsubstituted or are substituted by other than hydrogen at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by one or more suitable groups (which may be the same or different). Optional substitution is also indicated by the phrase "substituted with from 0 to X substituents," where X is the maximum number of possible substituents. Certain optionally substituted groups are substituted with from 0 to 2, 3 or 4 independently selected substituents (i.e , are unsubstituted or substituted with up to the recited maximum number of substituents). "B1," as used herein, refers to the human B1 bradykinin receptor reported by Menke ei al.
(1994) J. Biol Chem. 2(59:21583-21586, as well as allelic variants thereof and homologues thereof found in other species (e.g., GenBank Accession Number AAX 14712 (Macaca fascicular is)).
The term "B| agonist" refers to a compound that binds B1 and induces signal transduction mediated by B1. Bi agonists include, for example, bradykinin and kallidin (lysyl-bradykinin), as well as peptide portions or variants of bradykinin or kallidin that bind B; and retain activity. Representative Bs agonists include, but are not limited to. desArg9bradykinin and desArg10kallidin.
A "B] antagonist" is a compound that detectably inhibits signal transduction mediated by Bj. Such inhibition may be determined using the representative calcium mobilization assay provided in Example 7. Preferred Bi antagonists have an IC50 Of 5 μM or less in this assay, more preferably 2 μM or less, and stili more preferably 1 μM or less, 500 nM or less, 100 iiM or less or 10 nM or less. In certain embodiments, the B) antagonist is specific for Bi (i.e., the 3C50 value in a similar assay performed using the Bj receptor is greater than 2 μM and/or the IC50 ratio (B7/B1) is at least 10, preferably 100, and more preferably at least 1000). Bj antagonists preferably have minimal agonist activity (i.e., induce an increase in the basal activity of Bj that is less than 5% of the increase that would be induced by one EC50 of the peptide agonist desArg'°kaliidin, and more preferably have no detectable agonist activity within the assay described in Example 7). B1 antagonists for use as described herein are generally non-toxic. B; antagonists include neutral antagonists and inverse agonists.
A "neutral antagonist" of B1 is a compound that inhibits the activity of B1 agonist (e.g , desArg10kaIlidin) at Bj, but does not significantly change the basal activity of the receptor (i.e., within a calcium mobilization assay as described in Example 7 performed in the absence of agonist. Bi activity is reduced by no more than 10%, more preferably by no more than 5%. and even more preferably by no more than 2%; most preferably, there is no detectable reduction in activity). Neutral antagonists may. but need not. also inhibit the binding of agonist to Bi. An "inverse agonist" of Bi is a compound that reduces the activity of Bi below its basal activity level in the absence of actuating concentrations of agonist. Inverse agonists may also inhibit the activity of agonist at Bi, and/or may inhibit binding of Bj agonist to Bi. The reduction in basal activity of B, produced by an inverse agonist may be determined from a calcium mobilization assay, such as the assay of Example 7.
A "therapeutically effective amount" (or dose) is an amount that, upon administration to a patient, results in a discernible patient benefit (e.g., provides detectable relief from a condition being treated). Such relief may be detected using any appropriate criteria. A therapeutically effective amount or dose generally results in a concentration of compound in a body fluid (such as blood, piasma, serum, CSF, synovial fluid, lymph, cellular interstitial fluid, tears or urine) that is sufficient to result in detectable alteration in Brmediated signal transduction (using an assay provided herein). The discernible patient benefit may be apparent after administration of a single dose, or may become apparent following repeated administration of the therapeutically effective dose according to a predetermined regimen, depending upon the indication for which the compound is administered. For the treatment of pain, a discernible patient benefit is generally apparent after administration of a single therapeutically effective dose, although further benefit may become apparent following repeated administrations. A "patient" is any individual treated with a benzimidazole carboxamide derivative as provided herein. Patients include humans, as well as other animals such as companion animals (e.g., dogs and cats) and livestock. Patients may be experiencing one or more symptoms of a condition responsive to Bi modulation or may be free of such symptom(s) (i.e., treatment may be prophylactic in a patient considered to be at risk for the development of such symptoms). BENZIMIDAZOLE CARBOXAMIDE DERIVATIVES
As noted above, the present invention provides benzimidazole carboxamide derivatives of Formula I that may be used in a variety of contexts, including in the treatment of conditions responsive to Bj modulation, as described herein.
Formula 1
Figure imgf000011_0001
Such compounds may also be used within in vitro assays (e.g., assays for Bi activity), as probes for detection and localization of B, and within assays to identify other B1 antagonists.
Within certain benzimidazole carboxamide derivatives of Formula 1, the variables A, B, D and E are each CH. Such compounds satisfy Formula ϊa:
Formula Ia
Figure imgf000011_0002
It will be apparent that the substitution of a ring carbon atom in Formula I or Ia with a substltuent represented by R| will result in the loss of the hydrogen atom at that ring carbon. Within certain embodiments, Ri represents from 0 to 2 substituents independently chosen from halogen, cyano and C1-C^alkyl.
The variable represented by R2 is, within certain embodiments, phenylQ-C^alky] that is substituted with from 0 to 3 substituents independently chosen from halogen, hydroxy, cyano, Q- Qaikyl, and Q-Qalkoxy. Representative such R2 moieties include, for example, benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, Q-C4alkyl, and C1- C4alkoxy. Within other embodiments, the variable represented by R2 is (4- to 7-membered heterocyc!oa!kyl)Q-C1aikyf that is substituted with from 0 to 3 substituents independently chosen from 0x0 and Q-C4alkyl.
Within the variable designated "Y," the sum of r and p ranges from 1 to 6 for certain benzimidazole carboxamide derivatives. Within certain such compounds, p is not zero. Certain Y groups satisfy the formula (CH2)rO-(CH2)p, which is substituted with from 0 to 4 substituents independently chosen from amino, hydroxy, cyano, Q-Cealkyl, Ci-Cealkenyl. C2-C6alkynyl, Q- Cδhaloalkyl and substituents of the same carbon atom or adjacent carbon atoms that are taken together to form CrCf,cycloaIkyl. Representative such Y groups include, for example, -O-CH2-, -CH2-O- CH2- -0-CH2-CH2- -CH2-O-CH2-CH2- and -CH2-CH2-O-CH2-. Other Y groups satisfy the formula -N(R10)-CH2-. Yet another Y group is -CH=CH-.
Within other benzimidazole carboxamide derivatives of Formula 1, Y is a group of the formula (CH2V(CH2),, that is substituted with from 0 to 4 substituents independently chosen from amino, hydroxy, cyano, C1-Cβalkyl. Ci-C6alkenyl, C2-C6alkynyl, Q-Cήhaloalkyl and substituents of the same carbon atom or adjacent carbon atoms that are taken together to form CVC6cycloaikyl. Representative such Y groups include, for example, Q-Qalkylene groups such as methylene and ethylene, each of which is optionally substituted as indicated above (e.g., unsubstituted or substituted with amino, hydroxy, cyano, Q -Chalky! (e.g., methyl or ethyl), C2-C4alkenyl or Q-dhaloalkyl).
Within certain benzimidazole carboxamide derivatives of Formulas 1 and Ja, R1 is hydrogen or Q-Qalkyl. Within certain other benzimidazoie carboxamide derivatives of Formula 1, Y is a group of the formula (CHJrCHRm-. wherein RiU is taken together with Rj to form a 4- to 7-rncrnbcrcd heterocycloalkyl. Certain such compounds further satisfy Formula II or Ha:
Figure imgf000012_0001
Figure imgf000012_0002
wherein m is 0, 1. 2 or 3 and the remaining variables are as described for Formula I.
Within certain benzimidazole carboxamide derivatives provided herein, the variables R4 and K5 are taken together to form a 4- to 7-membered heterocycloafkyl that is substituted with mono- or di-(CrC6alkyi)aminoCo-C4alkyl, (4- to 7-membered heterocycloa!kyl)Co-C}alkyl, ρheny[C0-C4alkyl or (6-membered heteroaryl)Co-C4aIkyl, each of which is unsubstituted or substituted with oxo, C,- C4alkyl or (5- or 6-membered heterocycIe)Co-C2alkyI. Certain such compounds further satisfy Formula III or Ilia:
Figure imgf000013_0001
wherein Z is N or CH; and R6 is piperidinylQrGtalkyl, pyrroϊidinylCo-Qaikyl, pyridmylC0-C4alkyl, pyrimidinyiC0-C4aIkyl, trtrahydropyrimidinylCo^alkyl, imidazolylCo-C4alkyl, dihydroimidazoiylCo- C4alkyl, thiazolylCo-C4alkyl, oxadiazolylCo-C4alkyl, phenyiCo-C4alkyI, or di-(C1-C4alkyl)aminoC0- C4alkyl, each of which is unsubstituted or substituted with one or two C1-C4alkyl. Within further such compounds:
R] represents from 0 to 2 substituents independently chosen from halogen, cyano, and C1-C4alkyl; R.2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen,
C]-C4alkyl, and C1-C4aikoxy: R3 is hydrogen or C1^alkyl; and Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
Within other benzimidazole carboxamide derivatives provided herein, R4 is CrC4alkyl; and R5 is phenylCo-C2aIkyl that is substituted with mono- or di-(C1-C6alkyI)aminoC0-C4aIkyl, (4- to 7- membered heterocycloalkyl)C0-C4alkyl, phenylC0-C4alkyl or (6-membered heteroaryl)C0-C4aIkyl, each of which is unsubstituted or substituted with oxo, C1-C4alkyl, (5- or 6-membered heteiOcycle)C0- C^alkyl, or mono- or di-(C]-Q,alky!)aminoCo-Qaikyl. Certain such compounds further satisfy Formula IV or IVa:
Figure imgf000013_0002
wherein R7 is methyl or ethyl; and Rs is piρeridinylC0-C4aIkyl, pyrrolidiny[C[rC4alkyI, pyridinylC0- C4alkyl, pyrimidinylCo-C4aIky[, trtrahydropyrimidinylCo-C^alkyl. imidazolyiC0-C4alkyl, dihydroimidazolylCo-C4alkyl, thiazolylC0-C4aIkyl, oxadiazolylCo-C4alkyl, phenylCo-C4aikyl, or di- (C1-C4alkyl)aminoC0-C4alkyl. each of which is unsubstituted or substituted with one or two C] - C4alkyl, Within further such compounds: R| represents from 0 to 2 substituents independently chosen from halogen, cyano. and C1-C4aIkyl;
R2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, C1 -C4alkyl, and C]-C4alkoxy; R3 is hydrogen or CrQalkyl; and Y is methylene or ethylene, each of which is un substituted or substituted with methyl or ethyl.
Within still further benzimidazole carboxamide derivatives provided herein, R4 and R5 are taken together to form a 8- to 14-membered spiro heterocycloaikyl that is unsubstituted or substituted with C1-C4alkyl, (C1-C7cyc]oalkyl)C0-C2alkyl or (5- or 6-membered heterocycle)C0"C2alkyL Certain such compounds further satisfy Formula V or Va:
Figure imgf000014_0001
wherein each n is independently 0 or 1 ; X is O or NR9; and R9 is CrC4a!kyl or (C3-C7cycϊoalkyl)Co- C2alkyl. Within further such compounds:
Ri represents from 0 Io 2 substituents independently chosen from halogen, cyano. and CrC4alkyl; R2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen,
CpQaikyl, and C1-C4alkoxy; R3 is hydrogen or C]-C4a]kyϊ; and Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
Representative benzimidazole carboxamide derivatives provided herein include, but are not limited to, those specifically described in the Examples below. It will be apparent that the specific compounds recited herein are representative only, and are not intended to limit the scope of the present invention. Fuilher, as noted above, all compounds of the present invention may be present as a free acid or base or as a pharmaceutically acceptable salt, hydrate or ester.
As noted above, within certain aspects, benzimidazole carboxamide derivatives provided herein are Bj modulators. In addition, certain benzimidazole carboxamide derivatives provided herein are specific for B] . B) modulator activity may be confirmed using a calcium mobilization aεsav such as the assa^ described in EXiimnls 7 herein
If desired. B1 binding activity of the benzimidazole carboxamide derivatives provided herein may be confirmed using the representative assay described in Example 6, herein, or using an assay described by Fox et al. (2005) Br. J. Pharmacol. 144:889-99. Preferred B, modulators exhibit a K1 within such an assay of 5 micromolar or less, more preferably 2 micromolar or less, 1 rnicromolar or less, 500 nanomolar or less, 100 nanomolar or less or 10 nanomolar or less.
In vivo activity of B] modulators provided herein may be confirmed using any of a variety of animal models including, but not limited to, those described in the following documents (each of which is hereby incorporated by reference for its disclosure of the recited animal model):
Wood et al. (2003) J. Med. Chem. -/(5: 1803-06 - carrageenan-induced mechanical pressure hyperalgesia; Conley et al. (2005) Eur. J. Pharmacol. 527:44-51 - thermal antinociception and carrageenan-indυced mechanical pressure hypersensitivity;
Gougat et al. (2004) J. Pharmacol. Exper. Therap. 309:661-669 - UV irradiation-induced thermal hyperalgesia and chronic constriction of the sciatic nerve-induced neuropathy and in ischemia- induced injury;
Fox et al. (2005) Br. J. Pharmacol. 144:SS9-99 - complete Freund's adjuvant- induced mechanical pressure hypersensitivity; Gabra et ai. (2005) J. Neuropathol. Exp. Neurol. 64:782-89 - diabetes-induced peripheral neuropathic pain; Pesquero et al. (2000) Proc. Nail. Acad. ScL USA 97:% 140-45 - carrageenan-induced pleurisy
(inflammation around the lung);
Lawson et al. (2005) Eur. J. Pharmacol . 514:69-78 - diabetes-induced vascular disease; Hirata et a (2003) Eur. J. Pharmacol. 474:255-60 - ACE inhibitor-induced cough; Mazzuferi et al. (2005) Neuroscience. 135:979-86 - neuronal hyperexcitability in epilepsy. If desired, compounds provided herein may be evaluated for certain pharmacological properties including, but not limited to, oral bioavailability (preferred compounds are orally bioavailabie to an extent allowing for therapeutically effective doses of less than 140 mg/kg, preferably less than 50 mg/kg, more preferably less than 30 mg/kg, even more preferably less than 10 mg/kg, still more preferably less than 1 mg/kg and most preferably less than 0.1 mg/kg), toxicity (a preferred compound is nontoxic when a therapeutically effective amount is administered to a subject), side effects (a preferred compound produces side effects comparable Io placebo when a therapeutically effective amount of the compound is administered to a subject), serum protein binding and in vitro and in vivo half-fife (a preferred compound exhibits an in vivo half-life allowing for Q.3. D. dosing, preferably T. I. D, dosing, more preferably B. LD. dosing, and most preferably oπce-a- day dosing). In addition, differential penetration of the blood brain barrier may be desirable. Routine assays that are well known in the art may be used to assess these properties, and identify superior corn pounds for a particular use. For example, assays used to predict bioavailability include transport across human intestinal cell monolayers, including Caco-2 cell monolayers. Penetration of the blood brain barrier of a compound in humans may be predicted from the brain levels of the compound in laboratory animals given the compound (e.g., intravenously). Serum protein binding may be predicted from albumin binding assays. Compound half-life is inversely proportional to the frequency of dosage of a compound. In vitro half-lives of compounds may be predicted from assays of microsomal half-life as described herein.
As noted above, preferred compounds provided herein are nontoxic. In general, the term "nontoxic" as used herein shall be understood in a relative sense and is intended to refer to any substance that has been approved by the United States Food and Drug Administration ("FDA") for administration to mammals (preferably humans) or, in keeping with established criteria, is susceptible to approval by the FDA for administration to mammals (preferably humans). In addition, a highly preferred nontoxic compound generaily satisfies one or more of the following criteria: (1 ) does not substantially inhibit cellular ATP production; (2) does not significantly prolong heart QT intervals; (3) does not cause substantial liver enlargement, or (4) does not cause substantia! release of liver enzymes.
As used herein, a compound that does not substantially inhibit cellular ATP production is a compound that satisfies the criteria set forth in Example 8, herein. In other words, cells treated as described in Example 8 with 100 μM of such a compound exhibit ATP levels that are at least 50% of the ATP levels detected in untreated cells. In more highly preferred embodiments, such cells exhibit ATP levels that are at least 80% of the ATP levels detected in untreated cells.
A compound that does not significantly prolong heart QT intervals is a compound that does not result in a statistically significant prolongation of heart QT intervals (as determined by electrocardiography) in guinea pigs, minipigs or dogs upon administration of a dose that yields a serum concentration equal to the EC50 or IC10 for the compound. In certain preferred embodiments, a dose of 0.01 , 0.05, 0.1 , 0.5, 1, 5, 10, 40 or 50 mg/kg administered parenterally or orally does not result in a statistically significant prolongation of heart QT intervals. By "statistically significant" is meant results varying from control at the p<0.I level or more preferably at the p<0.05 level of significance as measured using a standard parametric assay of statistical significance such as a student's T test.
A compound does not cause substantial liver enlargement if daily treatment of laboratory rodents (e.g., mice or rats) for 5-10 days with a dose that yields a serum concentration equal to the
EC50 or IC50 for the compound results in an increase in liver to body weight ratio that is no more than
100% over matched controls. Jn more highly preferred embodiments, such doses do not cause liver enlargement of more than 75% or 50% over matched controls. If non-rodent mammals (e.g., dogs) are used, such doses should not result in an increase of liver to body weight ratio of more than 50%. preferably not more than 25%, and more preferably not more than 10% over matched untreated controls. Preferred doses within such assays include 0.01. 0.05. 0.1. 0.5. 1. 5. 10, 40 or 50 mg/kg αuπiiiiistei ed pώiciitei aϊl} Oi orally.
Similarly, a compound does not promote substantial release of liver enzymes if administration of twice the minimum dose that yields a serum concentration equal to the EC50 or 1C™ for the compound does not elevate serum levels of ALT, LDH or AST in laboratory rodents by more than
100% over matched mock-treated controls. In more highly preferred embodiments, such doses do not elevate such serum levels by more than 75% or 50% over matched controls. Alternatively, a compound does not promote substantial release of liver enzymes if, in an in vilrυ hepatocyte assay, concentrations (in culture media or other such solutions that are contacted and incubated with hepatocytes in vitro) that are equal to the EC50 or IC50 for the compound do not cause detectable release of any of such liver enzymes into culture medium above baseline levels seen in media from matched mock-treated controi cells. In more highly preferred embodiments, there is no detectable release of any of such liver enzymes into culture medium above baseline levels when such compound concentrations are five-fold, and preferably ten-fold the EC50 or IC50 for the compound.
In other embodiments, certain preferred compounds do not inhibit or induce microsomal cytochrome P450 enzyme activities, such as CYP 1A2 activity. CYP2A6 activity, CYP2C9 activity, CYP2C19 activity, CYP2D6 activity, CYP2E1 activity or CYP3A4 activity at a concentration equal to the EC50 or IC50 for the compound.
Certain preferred compounds are not clastogenic (e.g., as determined using a mouse erythrocyte precursor cell micronucleus assay, an Ames raicronucleus assay, a spira! micronucleus assay or the like) at a concentration equal the EC50 or IC50 for the compound. In other embodiments, certain preferred compounds do not induce sister chromatid exchange (e.g., in Chinese hamster ovary cells) at such concentrations.
For detection purposes, as discussed in more detail below, compounds provided herein may be isotopically-Iabeled or radiolabeled. For example, such compounds may have one or more atoms replaced by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be present in the compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H, 3H, 11C, '3C, '4C, '5N, 18O, 17O5 31P, '2P, '5S, 18F and 36Cl. In addition, substitution with heavy isotopes such as deuterium (i.e., 2H) can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances.
PREPARATION OF BENZIMIDAZOLE CARBOXAMIDE DERIVATIVES
Compounds provided herein may generally be prepared using standard synthetic methods. In general, starting materials are commercially available from suppliers such as Sigma-Aldrich Corp. (St.
Louis, MO), or may be synthesized from commercially available precursors using established protocols. By way of example, a synthetic route similar to that shown in any of the following
Schemes may be used, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon appreciated by those skilled in the art. It will be apparent that the reagents and synthetic transformations in the following Schemes and in the art can be readily modified to produce additional compounds of Formula I. Each variable in the following Schemes refers to any group consistent with the description of the compounds provided herein.
When a protecting group is required, an optional deprotection step may be employed. Suitable protecting groups and methodology for protection and deprotection, such as those described in Protecting Groups in Organic Synthesis by T. Greene, are well known. Compounds and intermediates requiring protection/deprotection will be readily apparent. Certain abbreviations used in the following Schemes and in the Examples include:
BOP benzotriazol-1 -yl-oxy-ιris(dimethylamino)phosphonium hexafluorophosphate
DIEA N,N-diisopropylethylamine DMC 2-chloi*o-K3-dimethy]imidazoHdinium chloride
DMF dimethylformamide
DMSO dimethylsulfoxide
Eq. equivalent(s)
EtOAc ethyl acetate h hour(s)
1H NMR proton nuclear magnetic resonance
LC-MS liquid chromatography/mass spectrometry
MeOH methanol
MHz megahertz
M+l mass + 1 mm minute(s)
MS mass spectrometry δ chemicai shift rt room temperature
PTLC preparative thin layer chromatography tBuOK potassium tø7-bυtoxide
TFA trifluoroacetic acid
Figure imgf000018_0001
[ 7 Scheme 2
Figure imgf000019_0001
In certain embodiments, a compound provided herein may contain one or more asymmetric carbon atoms, so that the compound can exist in different stereoisomers forms. Such forms can be, for example, racemates or optically active forms. As noted above, all stereoisomers are encompassed by the present invention. Nonetheless, it may be desirable to obtain single enantiomers (i.e., optically active forms). Standard methods for preparing single enantiomers include asymmetric synthesis and 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.
Compounds may be radiolabeled by carrying out their synthesis using precursors comprising at least one atom that is a radioisotope. Each radioisotope is preferably carbon (e.g., 14C), hydrogen (e.g., 3I-I), sulfur (e.g., 33S) or iodine (e.g., 125I). Tritium labeled compounds may also be prepared catalytically via platinum-catalyzed exchange in tritiated acetic acid, acid-catalyzed exchange in tritiated trifluoroacetic acid, or heterogeneous-catalyzed exchange with tritium gas using the compound as substrate, in addition, certain precursors may be subjected to tritium-halogen exchange with tritium gas, tritium gas reduction of unsaturated bonds, or reduction using sodium borotritide, as appropriate. Preparation of radiolabeled compounds may be conveniently performed by a radioisotope supplier specializing in custom synthesis of radiolabeled probe compounds. PHARMACEUTICAL COMPOSITIONS
The present invention also provides pharmaceutical compositions comprising one or more benzimidazole carboxamide derivatives provided herein, together with at least one physiologically acceptable carrier or excipient. Pharmaceutical compositions may comprise, for example, one or more of water, buffers (e.g., neutral buffered saline or phosphate buffered saline), ethanol, mineral oil. vegetable oil, dimethylsulfoxide, carbohydrates (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. In addition, other active ingredients may (but need not) be included in the pharmaceutical compositions provided herein.
Pharmaceutical compositions may be formulated for any appropriate manner of administration, including, for example, topical, oral (including, but not limited to, sublingual), nasal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions suitable for oral use are preferred. Such compositions 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 of the present invention may be formulated as a lyophilizate.
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 (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents (e.g., corn starch or alginic acid), binding agents (e.g., starch, gelatin or acacia) and lubricating agents (e.g., magnesium stearate, stearic acid or talc). The tablets may be un coated 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 (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 (e.g., peanut oil, liquid paraffin or olive oil).
Aqueous suspensions coiitd.ni the sctive iiidtertiil(s) in admixture with excipienls suitable foi the manufacture of aqueous suspensions. Such excipients include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanlh and gum acacia); and dispersing or wetting agents (e.g., naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with fatty acids such as pol>oxyethylene stearate. condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyieneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyetbyleπe 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, such as 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 ingredient(s) in a vegetable oil {e.g., arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide palatable oral preparations. Such suspensions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.
Pharmaceutical compositions may also be formulated as oil-in-water emulsions. The oily phase may be a vegetable oil (e.g., olive oil or arachis oil), a mineral oil {e.g., liquid paraffin) or a mixture thereof. Suitable emulsifying agents include naturally-occurring gums (e.g., gum acacia or gum tragacanth). naturally- occurring phosphatides (e.g., soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol), anhydrides (e.g., sorbitan monoleate) and condensation products of partial esters derived from fatty acids and hexitol with ethylene oxide (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. 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 (e.g., ethanol or isopropyl alcohol) or glycerin; glycols (e.g., butylene, isoprene or propylene glycol); aliphatic alcohols (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, acylgiycerols (including oils, such as 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 and emulsions. 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.
A pharmaceutical composition may be prepared as a sterile injectible aqueous or oleaginous suspension. The compound(s) provided herein, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Such a composition may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents such as those mentioned above. Among the acceptable vehicles and solvents that may be employed are water, 1 ,3-butanediol,
Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectible compositions, and adjuvants such as local anesthetics, preservatives and/or buffering agents can be dissolved in the vehicle.
Compounds may also be formulated as suppositories (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.
Pharmaceutical compositions may be formulated for release at a pre-detcrmined rate. iiiMdiitdiieuus i elcdSe ificty oe dLπievcu, foi example, vid Sublingual administration (i.e., administration by mouth in such a way that the active ingredient(s) are rapidly absorbed via the blood vessels under the tongue rather than via the digestive tract). Controlled release formulations (/ e., formulations such as a capsule, tablet or coated tablet that slows and/or delays release of active ingredient(s) following administration) may be administered by. for example, oral, rectal or subcutaneous implantation, or by implantation at a target site. In general, a controlled release formulation comprises a matrix and/or coating that delays disintegration and absorption in the gastrointestinal tract (or implantation site) and thereby provides a delayed action or a sustained action over a longer period. One type of controlled-release formulation is a sustained-release formulation, in which at least one active ingredient is continuously released over a period of time at a constant rate. Preferably, the therapeutic agent is released at such a rate that blood (e g., plasma) concentrations are maintained within the therapeutic range, but below toxic levels, over a period of time that is at least 4 hours, preferably at least 8 hours, and more preferably at least 12 hours. 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.
Controlled release may be achieved by combining the active ingredient(s) with a matrix material that itself alters release rate and/or through the use of a controlled-release coating. The release rate can be varied using methods well known in the ait, including (a) varying the thickness or composition of coating, (b) altering the amount or manner of addition of plasticizer in a coating, (c) including additional ingredients, such as release-modifying agents, (d) altering the composition, particle size or particle shape of the matrix, and (e) providing one or more passageways through the coating. The amount of modulator contained within a sustained release formulation depends upon, for example, the method of administration (e.g., the site of implantation), the rate and expected duration of release and the nature of the condition to be treated or prevented.
The matrix material, which itself may or may not serve a controlled-release function, is generally any material that supports the active ingredient(s). For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. Active ingredient(s) may be combined with matrix material prior to formation of the dosage form (e.g., a tablet). Alternatively, or in addition, active ingredient(s) may be coated on the surface of a particle, granule, sphere, microsphere, bead or pellet that comprises the matrix material. Such coating may be achieved by conventional means, such as by dissolving the active ingredient(s) in water or other suitable solvent and spraying. Optionally, additional ingredients are added prior to coating (e.g., to assist binding of the active ingredient(s) to the matrix material or to color the solution). The matrix may then be coated with a barrier agent prior to application of controlled-releasc coating. Multiple coated matrix units may, if desired, be encapsulated to generate the final dosage form.
In certain embodiments, a controlled release is achieved through the use of a controlled release coating (i.e., a coating that permits release of active ingredient(s) at a controlled rate in aqueous medium). The controlled release coating should be a strong, continuous film that is smooth, capable of supporting pigments and other additives, non-toxic, inert and tack-free. Coatings that regulate release of the modulator include pH-independent coatings, pH-dependent coatings (which may be used to release modulator in the stomach) and enteric coatings (which allow the formulation to pass intact through the stomach and into the small intestine, where the coating dissolves and the contents are absorbed by the body), it will be apparent that multiple coatings may be employed (e.g., to allow release of a portion of the dose in the stomach and a portion further along the gastrointestinal tract). For example, a portion of active ingredient(s) may be coated over an enteric coating, and thereby released in the stomach, while the remainder of active ingredieni(s) in the matrix core is protected by the enteric coating and released further down the GI tract. pH dependent coatings include, for example, shellac, cellulose acetate phthalate, polyvinyl acetate phthaiate, hydroxypropylmelhylcellulose phthalate, methacrylϊc acid ester copolymers and zein.
In certain embodiments, the coating is a hydrophobic material, preferably used in an amount effective to slow the hydration of the gelling agent following administration. Suitable hydrophobic materials include alkyl celluloses (e.g., ethylcellulose or carboxymethylcellulose), cellulose ethers, cellulose esters, acrylic polymers (e.g., poly(acrylic acid), poly(methacrylic acid), acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxy ethyl methacrylates. cyanoelhyl methacrylate, methacrylic acid alkamide copolymer, po!y(methyl methacrylate), polyacry! amide, ammonio methacrylate copolymers, aminoalkyi methacrylate copolymer, poly(methacrylic acid anhydride) and glycidyl methacrylate copolymers) and mixtures of the foregoing. Representative aqueous dispersions of ethylcellulose include, for example, AQUACOATCg) (FMC Corp., Philadelphia, PA) and SURELEASE® (Coiorcon, Inc.. West Point, PA), both of which can be applied to the substrate according to the manufacturer's instructions. Representative acrylic polymers include, for example, the various EUDRAG1T® (Rohm America, Pjscatavvay, NJ) polymers, which may be used singly or in combination depending on the desired release profile, according to the manufacturer's instructions. The physical properties of coatings that comprise an aqueous dispersion of a hydrophobic material may be improved by the addition or one or more plasticizers. Suitable plasticizers for alkyl celluloses include, for example, dibutyl sebacate, diethyl phthalate, trielhyl citrate, tributyl citrate and triacetin. Suitable plasticizers for acrylic polymers include, for example, citric acid esters such as triethyl citrate and tributyl citrate, dibutyl phthalate, polyethylene glycols, propylene glycol, diethyl phthalate, castor oil and triacetin.
Controlled-release coatings are generally applied using conventional techniques, such as by spraying in the form of an aqueous dispersion. If desired, the cυating may cuiiipnse poics Oi uiduπeli to facilitate release of active ingredient. Pores and channels may be generated by well known methods, including the addition of organic or inorganic material that is dissolved, extracted or leached from the coating in the environment of use. Certain such pore-forming materials include hydrophilic polymers, such as hydroxyalkylcelluloses (e g., hydroxypropylmethylcellulose). cellulose ethers, synthetic water-soluble polymers (e.g., polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone and polyethylene oxide), water-soluble poljdextrose, saccharides and polysaccharides and alkali metal salts. Alternatively, or in addition, a controlled release coating may include one or more orifices, which may be formed my methods such as those described in US Patent Nos. 3.845,770: 4,034,758; 4,077,407; 4,088,864; 4.783,337 and 5,071.607. Controlled-release may also be achieved through the use of transdermal patches, using conventional technology (see, e.g , US Patent No. 4,668,232). Further examples of controlled release formulations, and components thereof, may be found, for example, in US Patent Nos. 4,572,833; 4,587, 1 17; 4,606,909; 4,610,870; 4,684,516; 4,777,049: 4,994,276; 4,996,058; 5, 128,143; 5,202,128; 5,376,384; 5,384,133; 5,445,829; 5,510, 1 19; 5,618,560; 5,643.604; 5,891 ,474; 5,958,456; 6,039,980; 6, 143,353; 6,126,969; 6,156,342; 6,197,347; 6,387,394; 6,399,096; 6,437,000; 6,447,796; 6,475,493; 6,491,950; 6,524,615; 6,838,094; 6,905.709; 6,923,984; 6,923,988; and 6,91 ] , 217; each of which is hereby incorporated by reference for its teaching of the preparation of controlled release dosage forms.
In addition to or together with the above modes of administration, a compound provided herein may be conveniently added to food or drinking water (e.g., for administration to non-human animals including companion animals (such as dogs and cats) and livestock). Animal feed and drinking water compositions may be formulated so that the animal takes in an appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to feed or drinking water.
Benzimidazole carboxamide derivatives provided herein are generally administered in a therapeutically effective amount. Preferred systemic doses are no higher than 50 mg per kilogram of body weight per day (e.g., ranging from about 0.001 mg to about 50 mg per kilogram of body weight per day), with oral doses generally being about 5-20 fold higher than intravenous doses (e.g., ranging from 0.01 to 40 mg per kilogram of body weight per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage unit will vary depending, for example, upon the patient being treated and the particular mode of administration. Dosage units will generaily contain from about 10 μg to about 500 mg of an active ingredient. Optimal dosages may be established using routine testing, and procedures that are well known in the art.
Pharmaceutical compositions provided herein may, but need not, further comprise one or more additional pharmaceutical agents, such as an anti-inflammatory agent or analgesic. Anti- inflammatory agents include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), nonspecific and cyclooxygenase-2 fCOX-2) specific cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, Icilunornide, cyclosporins A, IM gold, minocycline, azathioprine, tumor necrosis factor (TNF) receptor antagonists, soluble TNF alpha receptor (etanercept), anti~TNF alpha antibodies (e.g., infliximab and adaϋmumab), anti-C5 antibodies, interleukin-1 (IL-I) receptor antagonists (e.g., anakmra or IL-I trap), IL-18 binding protein, CTLA4-Ig (e.g., abatacept), anti- human IL-6 receptor monoclonal antibody (e.g., tocilizumab), LFA-3-ϊg fusion proteins (e.g., alefacept), LFA-I antagonists, anti-VLA4 monoantibody (e.g., natalizumab), anti-CD 1 I a monoclonal antibody, anti-CD20 monoclonal antibody (e g, rituximab), anti-IL-12 monoclonal antibody. anti-IL- 15 monoclonal antibody, CDP 484, CDP 870, chemokine receptor antagonists, selective iNOS inhibitors. p38 kinase inhibitors, integrin antagonists, angiogenesis inhibitors, and TMI-I dual inhibitors. Further anti-inflammatory agents include meloxicam, rofecoxib, celecoxib, etoricoxib. parecoxib. vaidecoxib and tilicoxib. NSAIDs include, but are not limited to, ibuprofen. flurbiprofen, naproxen or naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin. diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, keloprofen, sodium nabumetone, sulfasalazine, tolmetin sodium, and hydroxychloroquine. One class of NSAIDs consists of compounds that inhibit cyclooxygenase (COX) enzymes: such compounds include celecoxib and rofecoxib. NSAIDs further include salicylates such as acetylsalicylic acid or aspirin, sodium salicylate, choline and magnesium salicylates, and salsalate, as well as corticosteroids such as cortisone, dexamethasone. methylprednisolone, prednisolone, prednisolone sodium phosphate, and prednisone. Certain analgesics for use in combination with B| modulators provided herein are also antiinflammatory agents, and are listed above. Other such medications include narcotic agents which typically act at one or more opioid receptor subtypes (e.g , μ, K and/or δ), preferably as agonists or partiai agonists. Such agents include opiates, opiate derivatives and opioids, as well as pharmaceutically acceptable salts and hydrates thereof. Specific examples of narcotic analgesics include, within preferred embodiments, alfentanil, alphaprodme, anileridine, bezitramide, buprenorphine, butorphanol, codeine, diacetyldihydromorphine, diacetylmorphine, dihydrocodeine, diphenoxylate, ethylmorphine, fentanyl, heroin, hydrocodone, hydromorphone, isomethadone, levomethorphan, levorphane, levorphanol, meperidine, metazocine, methadone, methorphan, metopon, morphine, nalbuphine, opium extracts, opium fluid extracts, powdered opium, granulated opium, raw opium, tincture of opium, oxycodone, oxymorphone, paregoric, pentazocine, pethidine, phenazocine, piminodine, propoxyphene, racemethorphan, racemorphan, sulfentanyl, thebaine and pharmaceutically acceptable salts and hydrates of the foregoing agents.
Other examples of narcotic analgesic agents include acetorphine, acetyldihydrocodeme. acetyl methadol, ailylprodine, alphracetylmethadol, alphameprodine, alphamethadol, benzethidme, benzylmorphine, betacetylmethadol, betameprodme. betamethadol, betaprodinc, clonitazene, codeine methylbromide. codeine-N-oxide. cyprenorphine, desomorphine, dextromoramide. diampromide.
Figure imgf000026_0001
dihydiornoiphine, uirneaOλadol, diniepheptanol, dimctiiyltliitunubutciic. dioxaphetyl butyrate, dipipanone, drotebanol, ethanol, cthylmethylthiambutene. etonitazene, etorphine. etoxeridine, furethidine. hydromorphinol, hydrox\ pethidine, ketobcmidone, levomoramide. ievopheπacylraorphan. methyldesorphine. methyldihydromorphine, morpheridine. morphine methylpromide, morphine methylsulfonate, morphine-N-oxide. mjrophin, naloxone, naityhexone, nicocodeine. nicomorphine. noracymethadol. norlevorphanol. normethadone, normorphine, πorpipanone, pentazocaine, phenadoxone. phenampromide, phenomorphan. phenoperidine. piritramide. pholcodine. proheptazoine. properidine. propiran, racemoramide, thebacon. trimeperidine and the pharmaceutically acceptable salts and hydrates thereof.
Further specific representative analgesic agents include, for example acetaminophen (paracetamol): aspirin and other NSAΪDs described above; NR2B antagonists; capsaicin receptor antagonists: anti-migraine agents; anticonvulsants such as oxcarbazepine and carbarn azepine; antidepressants (such as TCAs, SSRIs. SNRIs, substance P antagonists, etc.); spinal blocks; pentazocine/naloxone; meperidine; Ievorphanol; buprenorphine; hydromorphone; fentanyl; sufentanyl; oxycodone; oxycodone/acetaminophen, nalbuphine and oxymorphone. Still further analgesic agents include CB2-receptor agonists, such as AM1241. capsaicin receptor antagonists and compounds that bind to the α2δ subunit of voltage-gated calcium channels, such as gabapentin and pregabalin.
Representative anti-migraine agents for use in combination with a B; modulator provided herein include CQRP antagonists, ergotamines and 5-HTj agonists, such as sumatripan, naratriptan, zolmatriptan and rizatriptan.
Pharmaceutical compositions may be packaged for treating conditions responsive to B] modulation (e.g., treatment of pain, inflammation or other disorder(s) recited herein). Packaged pharmaceutical preparations generally comprise a container holding a therapeutically effective amount of a pharmaceutical composition as described above and instructions (e.g., labeling) indicating that the composition is to be used for treating a condition responsive to B] modulation in a patient (e.g., pain or other disorder as indicated herein). In certain embodiments, a packaged pharmaceutical preparation comprises one or more benzimidazole carboxamide derivatives provided herein and one or more additional agents in the same package, either in separate containers within the package or in the same container (i.e., as a mixture). Preferred mixtures are formulated for oral administration (e.g., as pills, capsules, tablets or the like). In certain embodiments, the package comprises a label bearing indicia indicating that the components are to be taken together for the treatment of pain.
METHODS OF USE
Within certain aspects, the present invention provides methods for treating a condition responsive to B1 modulation in a patient. The patient may be afflicted with such a condition, or may be free of symptoms but considered at risk for developing such a condition. A condition is ''responsive to Bj modulation11 if the condition or symptom(s) thereof are alleviated, attenuated, delayed or otherwise improved by modulation of Bi activity. In general, such methods comprise administering to the patient a therapeutically effective amount of at least one benzimidazole carboxamide derivative as provided herein. Conditions responsive to Bi modulation include, for example pain; inflammation including neuroinflammation (such as atherosclerosis), inflammation associated with airway diseases (e.g., asthma, including allergic asthma, exercise-induced bronchoconstriction, occupational asthma, and other non-allergic asthmas), and inflammatory skin disorders (e.g., psoriasis and eczema)); respiratory disorders including bronchoconstriction. asthma, chronic obstructive pulmonary disease (e.g., emphysema), chronic cough (including ACE-inhibitor cough), adult respiratory distress syndrome, bronchitis, pneumonia, allergic rhinitis and vasomotor rhinitis; vascular edema (including diabetes- related vascular disease); and epilepsy. Other conditions responsive to Bi modulation include diabetes (e.g., type II or non insulin dependent, as well as diabetic vasculopathy, diabetic neuropathy, diabetic retinopathy, post capillary resistance and symptoms associated with insuiitis), seizure disorders (e.g., epilepsy), multiple sclerosis, liver disease, cardiovascular disorders (e.g., atherosclerosis, congestive heart failure and myocardial infarction), neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease) rheumatoid arthritis, infection, cancer, cranial trauma, rhinitis, septic shock, endotoxic and pancreatic shock, anaphylaxis, inflammatory bowel disease, irritable bowel syndrome, pancreatitis, cystitis, uveitis, vascular permeability, gingivitis, osteoporosis, benign prostatic hyperplasia, hyperactive bladder, cerebral edema, vasodilation, hypotension associated with sepsis, edema resulting from trauma associated with burns, sprains or fracture, cerebral edema, angiodema, Crohn's disease and ulcerative colitis. Bj modulators may also be used as smooth muscle relaxants for treating spasms of the gastrointestinal tract of uterus. In certain embodiments, the condition responsive to Bi modulation is pain or inflammation.
Pain that may be treated using the B1 modulators provided herein may be chronic or acute and includes, but is not limited to, peripheral nerve-mediated pain (especially neuropathic pain, such as pain due to diabetes, postherpetic neuralgia, nerve injury, vulvodynia, root avulsions, painful traumatic neuropathy and painful polyneuropathy). Compounds provided herein may also be used in the treatment of, for example, viscera! pain (e.g., pancreatitis, interstitial cystitis and renal colic), persistent hyperalgesia, inflammatory pain, repetitive motion pain, carpel tunnel syndrome, perioperative pain, algesia, oral neuropathic pain, toothache (dental pain), denture pain, ocular pain, postherpetic neuralgia, diabetic neuropathy, chemotherapy-induced neuropathy, reflex sympathetic dystrophy, trigeminal neuralgia, bone and joint pain (e.g., pain associated with osteoarthritis), rheumatoid arthritis, myofascial pain (e.g., muscular injury and fibromyalgia), Guillain-Barre syndrome, meralgia paresthetica, burning-mouth syndrome and/or pain associated with nerve and root damage, including as pain associated with peripheral nerve disorders (e.g., nerve entrapment and brachial plexus avulsions, amputation, peripheral neuropathies including bilateral peripheral neuropathy, tic douloureux, atypical facial pain, nerve root damage, and arachnoiditis). Additional neuropathic pain conditions include causalgia (reflex sympathetic dystrophy - RSD, secondary to injury of a peripheral nerve), neuritis (including, for example, sciatic neuritis, peripheral neuritis, polyneuritis, optic neuritis, postfebrile neuritis, migrating neuritis, segmental neuritis and Gombault's neuritis), neuronitis, neuralgias (e.g., those mentioned above, cervicobrachial neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngial neuralgia, migranous neuralgia, idiopathic neuralgia, intercostals neuralgia, mammary neuralgia, mandibular joint neuralgia, Morton's neuralgia, nasociliary neuralgia, occipital neuralgia, red neuralgia, Sluder's neuralgia, splenopalatine neuralgia, supraorbital neuralgia and vidian neuralgia), musculoskeletal pain, AIDS-related neuropathy, MS- related neuropathy, central nervous system pain (e.g., pain due to brain stem damage, sciatica, and ankylosing spondylitis), and spinal pain, including spinal cord injury-related pain. Headache, including headaches involving peripheral nerve activity may also be treated as described herein. Such headache pain includes, for example, sinus, cluster (i.e., migranous neuralgia) and tension headaches, migraine, temporomandibular pain and maxillary sinus pain. For example, migraine headaches may be prevented by administration of a compound provided herein as soon as a pre-migrainous aura is experienced by the patient.
Further conditions that can be treated as described herein include Charcot's pains, intestinal gas pains, ear pain, heart pain, muscle pain, eye pain, orofacial pain (e.g., odontalgia), abdominal pain, gynaecological pain (e.g., menstrual pain, dysmenorrhoea, pain associated with cystitis, labor pain, chronic peϊvic pain, chronic prostitis and endometriosis), acute and chronic back pain (e.g., lower back pain), gout, scar pain, hemorrhoidal pain, dyspeptic pains, pain associated with angina, nerve root pain, "non-painful" neuropathies, complex regional pain syndrome, homotopic pain and heterotopic pain — including pain associated with carcinoma, often referred to as cancer pain (e.g., in patients with bone cancer), pain (and inflammation) associated with venom exposure (e.g., due to snake bite, spider bite, or insect sting) and trauma associated pain (e.g., post-surgical pain such as postmastectomy, postthoracotomy and stump pain; phantom limb pain, episiotomy pain, pain from cuts, musculoskeletal pain, bruises and broken bones, and burn pain, especially primary hyperalgesia associated therewith). Additional pain conditions that may be treated as described herein include pain associated with respiratory disorders as described above, autoimmune diseases, immunodeficiency disorders, hot flashes, inflammatory bowel disease, gastroesophageal reflux disease (GERD), irritable bowel syndrome and/or inflammatory bowel disease.
In certain embodiments, pain treated with B1 modulators provided herein is inflammatory pain, acute pain, dental pain, back pain, surgical pain, headache, neuropathic pain or pain from osteoarthritis or trauma.
It will be apparent that compounds provided herein may be administered alone or in combination with one or more additional agents that are suitable for treating the disorder of interest. Within such combination therapy, the compound(s) and additional agent(s) may be present in the same pharmaceutical composition, or may be aumπiisteied sepaiateiy in eithes ordei. Repieseπtatrve anti-inflammatory agents and analgesics for use in combination therapy include those indicated above. Within other aspects, B; modulators provided herein may be used within combination therapy for the treatment of conditions involving pain and/or inflammatory components. Such conditions include, for example, autoimmune disorders and pathologic autoimmune responses known to have an inflammatory component including, but not limited to, arthritis (especially rheumatoid arthritis), psoriasis, Crohn's disease, lupus erythematosus, irritable bowel syndrome, tissue graft rejection, and hyperacute rejection of transplanted organs. Other such conditions include trauma (e.g., injury to the head or spinal cord), cardio- and cerebrovascular disease and certain infectious diseases. Within such combination therapy, a Bi modulator is administered to a patient along with an additional analgesic and/or anti-inflammatory agent. The Bj modulator and additional analgesic and/or anti-inflammatory agent may be present in the same pharmaceutical composition, or may be administered separately in either order.
Administration to the patient can be by way of any means discussed above, including oral, topical, nasal or transdermal administration, or intravenous, intramuscular, subcutaneous, intrathecal, epidural, intracerebroveπtπ'cular or like injection. Oral administration is preferred in certain embodiments (e.g., formulated as pills, capsules, tablets or the like).
Treatment regimens may vary depending on the compound used and the particular condition to be treated. In general, a dosage regimen of 4 times daily or less is preferred, with 1 or 2 times daily particularly preferred. It will be understood, however, that the specific dose and treatment regimen 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 and diet of the patient the time of administration, the route of administration, the rate of excretion, any drug combination and the severity of the particular disease undergoing therapy. Dosages are generally as described above: in general, the use of the minimum dose sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using medical or veterinary criteria suitable for the condition being treated or prevented.
Suitable dosages for Bj modulators (either alone or within such combination therapy) are generally as described above. Dosages and methods of administration of any additional agent(s) (e.g., anti-inflammatory and/or analgesic agents) can be found, for example, in the manufacturer's instructions or in the Physician's Desk Reference. In certain embodiments, combination administration results in a reduction of the dosage of the additional agent required to produce a therapeutic effect (i.e., a decrease in the minimum therapeutically effective amount). Thus, preferably- the dosage of additional agent in a combination or combination treatment method of the invention is less than the maximum dose advised by the manufacturer for administration of the agent without combination with a compound of Formula 1. More preferably this dose is less than 3A, even more preferably less than 'Λ, and highly preferably less than ]A of the maximum dose, while most prefeiabh the dose is less than 10% υf the maximum uuse advised by the manufacturer for administration of the agcnt(s) when administered without combination administration as described herein. It will be apparent that the dose of compound as provided herein needed to achieve the desired effect may similarly be affected by the dose and potency of the additional agent.
Within separate aspects, the present invention provides a variety of non-pharmaceutical in vitro and in vivo uses for the compounds provided herein. For example, such compounds may be labeled and used as probes for the detection and localization of Bi (in samples such as cell preparations or tissue sections, preparations or fractions thereof). In addition, compounds provided herein that comprise a suitable reactive group (such as an aryl carbonyl. nitro or azide group) may be used in photoaffϊnity labeling studies of receptor binding sites. In addition, compounds provided herein may be used as positive controls in assays for receptor activity, as standards for determining the ability of a candidate agent to bind to B!, or as radiotracers for positron emission tomography (PET) imaging or for single photon emission computerized tomography (SPECT). Such methods can be used to characterize B1 receptors in living subjects. For example, a compound may be labeled using any of a variety of well known techniques (e.g., radiolabeled with a radionuclide such as tritium, as described herein), and incubated with a sample for a suitable incubation time (e.g., determined by first assaying a time course of binding). Following incubation, unbound compound is removed (e.g., by washing), and bound compound detected using any method suitable for the label employed (e.g., autoradiography or scintillation counting for radiolabeled compounds; spectroscopic methods may be used to detect luminescent groups and fluorescent groups). As a control, a matched sample containing labeled compound and a greater (e.g., 10-fold greater) amount of unlabeled compound may be processed in the same manner. A greater amount of detectable label remaining in the test sample than in the control indicates the presence of B] in the sample. Detection assays, including receptor autoradiography (receptor mapping) of B1 in cultured cells or tissue samples may be performed as described by Kuhar in sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998) John Wiley & Sons, New York.
The following Examples are offered by way of illustration and not by way of limitation. Unless otherwise specified all reagents and solvent are of standard commercial grade and are used without further purification. Using routine modifications, the starting materials may be varied and additional steps employed to produce other compounds provided herein.
EXAMPLES
Mass spectroscopy data in the following Examples is Electrospray MS, obtained in positive ion mode using a Micromass Time-of-Flight LCT (Micromass, Beverly MA), equipped with a Waters 600 pump (Waters Corp.; Milford, MA), Waters 996 photodiode array detector, and a Gilson 215 autosampler (Gilson, Inc.; Middleton, WI). MassLynx (Advanced Chemistry Development. Inc; Toronto, Canada) version 4.0 software with Open Lynx Global Server™, OpenLynx™ and AutoLynx™ processing is used for data collection and analysis. MS conditions are as follows: capillary voltage = 3.5 kV; cone voltage = 30 V, desolvation and source temperature = 35O0C and 12O0C, respectively; mass range = 181 -750 with a scan time of 0,22 seconds and an interscan delay of 0.05 min. Sample volume of 1 microliter is injected onto a 50x4.6mm Chromolith SpeedROD RP-18e column (Merck KGaA, Darmstadt, Germany), and eluted using a 2-phase linear gradient at a flow rate of 6 mf/min. Sample is detected using total absorbance count over the 220-340nm UV range. The eiution conditions are: Mobile Phase A - 95% water, 5% MeOH with 0.05% TFA; Mobile Phase B - 5% water, 95% MeOH with 0.025% TFA. The following gradient is used: 0-0.5 min 10-100%B, hold at 100%B to 1.2 min, return to 10%B at 1.21 min. Inject to inject cycle is 2.15 min. EXAMPLE 1. SYNTHESIS OF 1 -BENZYL-N-[2OXO-2-(4-PYRIDIN-4-YLPJPERAZIM-1 -YL)ETHYLJ- I H-
BENZ1MIDAZOLE-2-CARBOXAMIDE
Figure imgf000032_0001
STKP 1. PREPARATION OF 2- ΓRICΉLOROMETΠYL- 1 H-BENzOIMIDΛZOLE
Add methy l 2.2,2-ιrichioroacetimidaιe (31 mL, 0.25 moi) drop-wise to a solution of benzene- 1.2-diamine (25 g. 0.23 mol) in HOAc (300 mL) at 0 °C and allow the solution to warm to rt. After 2 h, pour the mixture onto 500 mL ice-H2O and stir for 1 h. Filter the solid and air-dry to give crude title compound.
STEP 2. PREPARATION OF 1 H-BENZOIMIDAZOL-2-CARBOXYLIC ACID MET HYL ESTER
Add Na2CO3 (9 g, 85 mmoi) to a solution of 2-tπchioromethyl-lH-benzoimidazole (20 g, 85 mmol) in 500 mL MeOH at it. Heat the mixture at reflux for 14 h and then cool to rt. Pour into 1 L H2O and filter off the solid. Evaporate the volatiles and extract the residue with EtOAc. Combine the extracts, dry over Na2Sθ4 and evaporate to give the title compound. STEP 3. PREPARATION OF 1 -BENZYL- 1H-BENZOIMIDAZOL-2-CARBOXYLIC ACID METHYL ESTER
Add tBuOK (2.02 g. 18 mmol) to a solution of lH-benzoimidazol-2-carboxyIic acid methyl ester (3 g, 17 mmol) in DMF (75 mL) and stir for 15 min at rt. Add benzyl chloride (800 mg, 18 mmol) in one portion. Heat the mixture at 50 0C for 5 h. Evaporate the solvent, add brine and extract with EtOAc. Dry over Na2SOj and remove the solvent to give crude title compound.
STEP 4. PREPARATION OF 1 -BENZYL- IH-BENZOJMIDAZOL^-CΛRBOXYLIC ACID
Add NaOH (3 mL of 10 N solution, 30 mmol) to a solution of l-benzyl-lH-benzoimidazol-2- carboxylic acid methyl ester (4.3 g, 16 mmol) in MeOH (10 mL) at rt. Heat the mixture at 50 °C for 4 h. Evaporate to dryness. Add H2O to dissolve the solid and adjust the pH Io 3 with 6 N HCl. Collect the solid by filtration and wash with H2O, ether and dry to give the title compound.
STEP 5. PREPARATION OF [( 1 -BENZYL-IH-BENZOIMIDAZOLE-CARBONYL)-AMINO]-ACET[C ACID METHYL ESTER
Add DMC (1 .3 g, 8 mmol) to a solution of 1 -benzyl- lH-benzoimidazol-2-carboxylic acid (1 g, 4 mmol), DIEA (2.4 mL, 14 mmol) and glycine methyl ester HCl salt (515 mg, 4.1 mmol) in 120 mL MeCN at rt. Stir the mixture for 2 h at rt. Purify by flash column (elute with 1 %MeOH in DCM) to give the title compound.
STEP 6. PREPARATION OF [(I -BENZYL- 1 H-BENZOIMIDAZOLE-CARBONYL)-AMINO]-ACETIC ACID
To NaOH (1 mL of ION solution; 10 mmol) add a solution of [( 1 -benzyl- 1 H-benzoimidazole- carbonyl)-amino]-acetic acid methyl ester (1.1 g, 3.4 mmol) in MeOH (5 mL). Heat the mixture at 50 0C for 4 h. Evaporate and add H2O to dissolve the solid and adjust the pH to 3 with 6 N HCl. Filter the solid and wash with H2O and ether and dry to give the title compound.
STEP 7. PREPARATION OF 1 -BENZYL-N-[2-OXO-2-(4-P YRIDIN-4- YLPIPERAZIN-1-YL)ETHYL]- IH- BENZIMIDAZ0LE-2-CARBOXAM1DE
Add DMC (33 mg, 0.2 nimol) to a solution of [(1-benzyl-IH-benzoimidazole-carbonyl)- amino "|-acetic acid (30 mg, 0.1 mmol), DIEA (61 μL, 0.35 mmol) and 1 -(4-pyridyl)-piperazine (18.3 mg, 0.1 mmol) in 0,5 mL MeCN at rt. Stir the mixture for 2 h at rt. Purify the crude product by flash column (elute with 1 % MeOH in DCM) to give the title compound. 1H NMR (CDCl3): 8.19-8.17 (d. 2H), 7.80-7.76 (d, I H), 7.54-7.52 (d, IH), 7.36-7.34 (m, 2H), 7.29-7.16 (m, 7H), 6.01 (s, 2H), 4.37 (s, 2H), 3.83-3.75 (m, 8H). EXAMPLE 2. SYNTHESIS OF N-(2-{[4-(4,5-DIHYDRO- l H-IMID.ΛZOL-2-
YL)BENZYL](METHYL)ΛMINO} -2-OXOETHYL)- l -(4-FLUOROBENZYL)- 1H-BENZIMIDΛZOLE-2- CARBOXAMIDE
Figure imgf000034_0001
S TEP l . PREPARATION OF [(I -BENZYL- IH-BENZOIMIDAZOLE^-CARBONYLVAMINOJ-ACETIC ACID
METHYL ESTER
Add tBuOK (68 mg. 0.6 mmol) to a solution of l H-benzoimidazol-2-carboxylic acid methyl ester (150 mg. 0.54 mmol) in DMF (3 mL) at rt and stir for 15 min. Add 4-fIuorobenzyl bromide (1 13 mg, 0.6 mmol) in one portion. Heat the mixture at 50 0C for 14 h. Evaporate, add brine and extract with EtOAc. Dry over Na2SO4 and remove the solvent Io give crude title compound.
STEP 2. PREPARATION OF [( 1 -BENZYL- 1 H-BENZOIMIDAZOLE^-CARBONYLJ-AMΪNΌJ-ACETIC ACID
Add NaOH (0.1 mL of l ϋ N solution, 1 mmol) to a solution oi [(I -benzyl- I H- benzoimidazole-2-carbonyl)-amino]-acetic acid methyl ester (50 mg. 0.13 mmol) in MeOH (1 mL) at rt. Heat the mixture at 50 0C for 4 h. Evaporate and add H^O to dissolve the solid and adjust the pH to 3 with 6 N HCl. Collect the solid by filtration and wash with H2O and ether and dry to give the title compound.
STEP 3. PREPARA TION OF N-(2-{ [4-(4,5-DIHYDRO- 1 H-IMIDAZOL-2-YL)BENZYL](METHYL) AMINO} -2-
OXOE π IYL)- 1 -(4-FLUOROBENZYL)-IH-BENZIMIDAZOLE-Z-CARBOXAMIDE
Add BOP (32 mg, 0.073 mmol) to a solution of [( I -benzyl- l H-benzoimidazole-2-carbony!)- amino]-acetic acid (16 mg, 0.049 mmol), [4-(4.5-dihydro-l H-imidazol-2-yI)-benzyl]-methylamine
(9.5 mg. 0.05 mmol). 1 -hydroxy benzotriazole (1 mg, 0.008 mmol) and LJlEA (20 μL, 0.12 mmol) in DMF (0.2 niL) at it. Stir the mixture for 14 h. Remove the solvent and purify by PTLC (elute with 10%MeOH/I%NH,OH in DCM) to give the title compound. 1H NMR (CD3OD): 7.84-7.66 (m, 4H), 7.56-7.48 (m, 2H), 7.41-7.26 (m, 4H), 7.08-7-6.92 (m, 2H), 6.0-5.97 (m, 2H), 4.82-4.75 (m, 2H), 4.42-4.35 (m, 2H), 4.10-4.89 (m, 4H), 3.10-3.05 (m, 3H).
EXAMPLE 3. ADDITIONAL REPRESENTATIVE BENZiMIDAZOLE CARBOXAMIDE DERfVATIVES
Using routine modifications, the starting materials may be varied and additional steps employed to produce other compounds provided herein. Compounds listed in Table I are prepared using such methods. A "*" in the column headed "IC50" indicates that the compound exhibits an IC50 (determined as described in Example 7) that is 5 micromolar or less. Mass spectroscopy data is provided as M+l in the column headed "MS," with the retention times ("Ret Time") given in minutes.
Table I Representative Benzimidazoie Carboxamide Derivatives
Figure imgf000035_0001
l-benzyl-N-[2-oxo-2- (4-ρyτidin-4- ylpiperazin-1 -yl)ethyl]- 455 2 1.1 1 H-benzimidazole-2- carboxamide
Figure imgf000035_0002
Figure imgf000036_0001
Ret
Figure imgf000037_0001
Figure imgf000038_0001
-
4892 11
Figure imgf000038_0002
Figure imgf000038_0003
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
1-(3-methylbutyl)-N-[2- oxo-2-(4-pyridin-4- ylpiperazιn-1-yl}ethylj- 435 2 1 2 1 H-benzfmidazole-2- carboxamide
N-ethyl-1-(4- methoxybeπzyl)-N-{2-
[4-(1 -methylpipendf n-
4-yl)piperazιn-1-ylj-2- 533 2 1.1 oxoethyl}-1 H- benzιmidazole-2- carboxamide
Figure imgf000042_0002
Figure imgf000043_0001
Ret
Figure imgf000044_0001
l-
5172 11
Figure imgf000044_0002
py I- - 5172 11
Figure imgf000044_0003
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
ReI
COMPOUND NAME MS Time IC3O
Figure imgf000048_0001
1-beπzyl-N-(2-{[4-(4,5- dihydro-1 H-ιmιdazol-2- yl)benzyl](methyl)amm o}-2-oxoeihyl)-N-ethyl- 509 3 1 0 1 H-benzιmιdazole-2- carboxamide
Figure imgf000048_0002
Figure imgf000048_0003
Ret
COMPOUND NAME MS Time IC50
Figure imgf000049_0001
Ret
Figure imgf000050_0001
Ret
COMPOUND NAME MS Time TC ,M
N-(2-{[4-{4 5-dshydro-
1 H-ιmιdazol-2- yl)benzyl]{methyl)amsn o}-2-oxoethyl)-1-(2- 504 3 1 0 morpholιπ-4-ylethyl}-
1 H-benzιmιdazoϊe-2- carboxamide
N-(2-{[4-(4 5-dιhydro-
1 H-ιmιdazo!-2- yl)benzyl](methyi)amiπ o}-2-oxoethyl)-1-(4- 499 2 1 1 fluorobenzyi)-1 H- benzιmιdazole-2- carboxairude
Figure imgf000051_0001
Figure imgf000051_0002
Ret
Figure imgf000052_0001
- 4693 12
4893 11
4893 11
Figure imgf000052_0002
Figure imgf000053_0001
Figure imgf000054_0001
Ret
Figure imgf000055_0001
- 469.2 1.1
489.1 1.2
Figure imgf000055_0002
yl-N~ -1- 489.3 1.1 -
Figure imgf000055_0003
1-benzyl-N-(2-{[4-<4,5- dihydro-1 H-imidazoi-2- yl)benzyl](methyi)amin o}-2-oxoethy!)-7- 495.2 1.1 methyi-1 H- benzfmidazole-2- carboxamide
Figure imgf000055_0004
Ret
COMPOUND NAME MS Tune ΪC50
N-(2-{[4-(4,5-dihydro-
1 H-inrndazol-2- yi)benzyl](methyl)amιn o}-2-oxoethyl)-1- 467 2 1 1 phenyf-1 H- benzιmrdazole-2- carboxamide
N-(2-{[4-(4,5-dιhydra-
1 H-ιmιdazol-2- yl)beπzyl](methy!)arτi!n o}-2-oxoethyl)-1-(2- 499 2 1 1 fluorobeπzyl)-1 H- beπzιmιdazole-2- carboxamide
1 -(3,5-dsfluorobenzyl)-
N-(2-{[4-(4,5-dιhydro-
1 H-ιmιdazol-2- yl)benzyl](methyl)amιn 517 2 1 1 o}-2-oxoethyi)-1 H- benzιmιdazole-2- carboxamide
Figure imgf000056_0001
2"
424 2 1 0
Figure imgf000056_0002
1-benzyl-N-(2-
{methyl[4~(1 ,4,5 6- tetrahydropyπnrndιn-2- y!)benzyl]amiπo}-2- 495 2 1 1 oxoethy!)~1 H- beπzιmιdazole-2- carboxamide
Figure imgf000056_0003
0
Figure imgf000057_0001
Ret
Figure imgf000058_0001
51
Figure imgf000059_0001
Figure imgf000060_0001
Ret
Figure imgf000061_0001
Figure imgf000062_0001
Ret MS Time IC50
486 1 0 85
504 1 0 7
504 1 0 5
461 1 0 57
Figure imgf000063_0001
dιn- H-
461 1 0 7
Figure imgf000063_0002
Figure imgf000064_0001
Ret
COMPOUND NAME MS Time IC 50
5112 089
Figure imgf000065_0001
Figure imgf000065_0002
Ret
Figure imgf000066_0001
Figure imgf000067_0001
EXAMPLE 4. ADDITIONAL REPRESENTATIVE BENZIMIDAZOLE CARBOXAMIDE DERIVATIVES
Using routine modifications, the starting materials may be varied and additional steps employed to produce other compounds provided herein. Compounds listed in Table II are prepared using such methods.
Table II Additional Representative Benzimidazole Carboxamide Derivatives
Figure imgf000067_0002
Figure imgf000068_0001
Figure imgf000069_0001
EXAMPLE 5. PREPARAI ION OF B1-TRAN SFECTED CELLS
This Example illustrates the preparation of Bptransfected cells for use in Bi binding and modulation assays (Examples 6 and 7).
Cynomolgus macaque lung total RNA is isolated as described by Chomzynski et al. ( 1987) Anal. Biυchem. /62: 156-159. A cDNA encoding Bi is cloned from the total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) with the following oligonucleotides:
Primer 1 : GGCGCT AGCCACC ATGGCATCCTGGCCCCCTC (SEQ ID NO: 1)
Primer 2: AGCCGTCCCAGATCTGAAC (SEQ ID NO:2)
Primer 3: GATCTGGGACGGCTTGGATG (SEQ ID NO:3)
Primer 4: CGGAGCTCTTAATTCCGCCAGAAAAGTTGGA (SEQ ID NO:4) Primer pairs 1 & 2 and 3 & 4 ace used to generate overlapping cDNA fragments corresponding to the entire protein coding sequence of cynomoigus macaque B1 cDNA are isolated and linked to form the fuli-Iength coding sequence (GenBank Accession Number AY788905). The construct is cloned into pcDNA 3.1 (Invitrogen. Carlsbad, CA) and transfecled into Chinese hamster ovary (CHO) cells using Lipofectamϊne (Invitrogen), resulting in cynomoigus macaque B 1-expressing CHO cells. Alternatively, the construct is cloned into pBAKPAK9 (Clontech, Mountain View, CA) and transfected into Sf9 cells to generate clonal baculovirus stocks. Clonal cell lines stably expressing the cynomoigus macaque B1 receptor are selected in G4I 8. A single clonal line that exhibits high levels of receptor expression is chosen for use in binding and calcium mobilization assays (Examples 6 and 7). Clonal baculovirus stocks are used to infect Sf9 cells such that the infect
Figure imgf000070_0001
d cells express high levels of recombinant B] receptors. These cells are used in radioligand binding assays (Example 6).
EXAMPLE 6. B1 RECEPTOR BINDING ASSAYS
This Example illustrates a representative Bi receptor binding assay that may be used to determine the binding affinity of compounds for B1.
A. [3 H]-DESARG 10KALLIDIN BINDIN TO INTACT IMR-90 CELLS OR CHO CELLS STABLY EXPRESSING
RAT B1
ΪMR-90 cells, which endogenously express human B5, are seeded into 24 well plates at
65.000 cells per well, cultured overnight, and then treated for 3 h with 0.2 ng/mL iπterleukin-l beta to induce B; expression (Mcnke, et al. (1994) J. Biol. Chem. 269:21583-86). CHO cells stably expressing rat B1 are seeded into 24 well plates at 200,000 cells per well and cultured overnight. The ceils are then washed 3 times with phosphate buffered saline (PBS). One hundred fifty microliters of binding buffer (50 mM Tris 7.4, 0.14 mg/mL, bacitracin, and 1 mg/mL BSA) is added to each well.
Various concentrations of test compound are added to each well from DMSO solutions such the final DMSO concentration is 1% by volume; some wells receive DMSO only, and some wells receiveDMSO plus 10μMdesArg10Kallidin to define non-specific binding. All wells then receive 0.3nM
(final concentration) [3H]-desArg10Kallidin. The plates are allowed to sit for 2 h at room temperature. Cells arc then washed three times, and lysed with 400 μl Ultima Gold scintillation fluid (PerkinElmer; Boston, MA; 20 min incubation). The fluid is then transferred to counting vials counted in a Packard liquid scintillation counter (PerkinElmer). The number of counts present in the scintillation fluid is plotted as a function of antagonist compound concentration and fitted to a logistical equation using SigmaPlot (Systat Software, Point Richmond, CA) to determine each compound's IC50 and K1 (e.g , as described by Szallasi. et al. (1993) J. Pharmacol Exp Ther. 25(5:678-83). B. [3H]-DESARD10KALLIDIN BINDING io MEMBRANE HOMOGENATES OF SF9 CELLS EXPRESSING
CYNOMOLGUS MACAQUE B 1
SiP cells infected with a baculovirus carrying the coding sequence for cynomolgus macaque Bi are harvested by centrilugation and frozen at -80 0C. Pellets are subsequently resuspended on ice in Tn s buffered saline (TBS: 50 mM Tris (pH 7.4). 120 mM NaCl). and cells are homogenized using a polytron for 30 seconds. The crude membrane fraction is collected by centrifugation at 20,000 ipm. Membranes are washed two times with TBS and collected by centrifugation each time. Protein content of the membranes is determined after the last wash and the concentration is adjusted to 0.7 μg/uL with binding buffer (50 mM Tris 7,4, 0.14 mg/mL bacitracin, and 1.0 mg/niL BSA). To perform the binding assay. 150 microliters of membrane fraction is added to each well of a 96-weli plate along with 50 μl [3H]-desArg'°Kallidm (0.3 nM final) and test compound in DMSO (final DMSO concentration = 1 %). Some wells receive DMSO only, and some welis receive DMSO plus 10 μM desArg10Kallidin to define non-specific binding. The 96 well plates are allowed to sit for 2 h at room temperature. Membrane proteins are then harvested by filtration onto GF/C fϊltermats (PerkinElmer) pre-soaked for 1 hr in 0.5 % polyethylenimine. After filtration, filters are dried and then counted in a Beta plate counter. The number of counts present in the scintillation fluid is plotted as a function of antagonist compound concentration and fitted to a logistical equation using SigmaPIot (Systat Software, Point Richmond, CA) to determine each compound's IC50 and K1 (e.g., as described by Szallasi, el al (1993) J. Pharmacol. Exp. Ther. 266:678-83).
EXAMPLE 7. CALCIUM MOBILIZATION ASSAY
This Example illustrates representative calcium mobilization assays for use in evaluating test compounds for agonist and antagonist activity.
Cynomolgus macaque Bj -express ing CHO cells (Example 5) are plated in a 96 well plate. The cells are cultured for 1 day, after which culture media is emptied from the plate and replaced with 50 μl of KRH (Krebs-Ringer HEPES buffer: 25 mM HEPES, 5 mM KCl, 0.96 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl3. 5 mM glucose. 1 mM probenecid. pH 7.4) supplemented with the calcium- sensitive fluorescent dye Fluo4-AM (5 μg/mi: Teflabs, Austin, TX). The cells are then incubated at 37 0C in an environment containing 5% CO2. After the 1 hour incubation, the dye solution is removed from the plate, the plate is washed once with KRH, and ] 00 μL KRH is added. DEI ERMINAT ION OF B 1 AGONIST EC50
100 μL KRH + 2% DMSO is added to each well of cells, such that the final volume in each well is 200 microliters and the final DMSO concentration is 1%. Various concentrations of the Bt agonist desArg10Kallidin are added. Addition of desArgl0Kallidin elicits a fluorescent response as consequence of increased intracellular calcium. This response is measured with a FLlPR instrument (Molecular Devices, Sunn>vale. CA) and determined to be desArg10KaI!idin concentration dependent. A plot of maximum fluorescent response as a function of desArg10KaIligin is generated and an EC50 (concentration required to elicit a 50% of maximal response) for the response is determined using the equation: y=a*(l/(l+(b/x)c)) in this equation, y is the maximum fluorescence signal, x is the concentration of the Bi agonist, a is the EmdX. b corresponds to the EC5-G value and c is the Hill coefficient.
By replacing the desArg10Kallidin with a test compound, this assay is also used to assess B| agonist activity of the test compound.
DETERMINATION OF ANTAGONIST ACTIVITY Various concentrations of test compounds are added to the cell plate prepared as described above in 100 μL KRH + 2% DMSO, such that the final volume in each well is 200 microliters and the final DMSO concentration is 1%. The EC50 concentration of desArg!°Kallidin is then added to each well of plates containing test compound to determine the extent to which each test compound inhibits an agonist-induced B1 response. The maximum fluorescent response is plotted as a function of test compound concentration in order to determine the IC50 (concentration required to inhibit 50% of the effect of agonist) for each compound at B1. Antagonists of Bj decrease this response by at least about 20%, preferably by at least about 50%. and most preferably by at least 80%, as compared to matched control (i.e , cells treated with desArg!°KaIlidin at the EC50 concentration in the absence of test compound), at a concentration of 10 micromoiar or less, preferably 1 micromolar or less. Alternatively, the data is analyzed as follows. First, the average maximum relative fluorescent unit (RFU) response from negative control wells (no agonist) is subtracted from the maximum response detected for each of the other experimental wells. Second, average maximum RFU response is calculated for the positive control wells (agonist wells). Then, percent inhibition for each compound tested is calculated using the equation:
Figure imgf000072_0001
The % inhibition data is plotted as a function of test compound concentration and test compound IC1o is determined using a linear regression in which x is ln(concenlration of test compound) and y is ln(percent inhibition/(100 - percent inhibition). Data with a percent inhibition that is greater than 90% or less than 15% are rejected and are not used in the regression. The IC50 is
^mtertiφt slope)
EXAMPLE S. MDCK CYTOTOXICITY ASSAY
This Example illustrates the evaluation of compound toxicity using a Madin Darby canine kidney (MDCK) cell cytotoxicity assay.
1 μL of test compound is added to each well of a clear bottom 96-well plate (Packard, Meriden, CT) to give final concentration of compound in the assay of 10 μM, 100 μM or 200 μM. Solvent without test compound is added to control wells.
MDCK cells. ATCC no. CCL-34 (American Type Culture Collection, Manassas, VA), are maintained in sterile conditions following the instructions in the ATCC production information sheet. Confluent MDCK ceils are trypsinized, harvested, and diluted to a concentration of 0.1 x 106 ceils/mL with warm (370C) medium (VITACELL Minimum Essential Medium Eagle, ATCC catalog # 30-
2003). 100 μL of diluted cells is added to each well, except for five standard curve control wells that contain 100 μL of warm medium without cells. The plate is then incubated at 370C under 95% O?,
5% CO2 for 2 hours with constant shaking. After incubation, 50 μL of mammalian cell lysis solution (from the Packard (Meriden, CT) ATP-LITE-M Luminescent ATP detection kit) is added per well, the wells are covered with PACKARD TOPSEAL stickers, and plates are shaken at approximately
700 rpm on a suitable shaker for 2 min.
Compounds causing toxicity will decrease ATP production, relative to untreated cells. The ATP-LITE-M Luminescent ATP detection kit is generally used according to the manufacturer's instructions to measure ATP production in treated and untreated MDCK cells. PACKARD ATP LITE-M reagents are allowed to equilibrate to room temperature. Once equilibrated, the lyophilized substrate solution is reconstituted in 5.5 mL of substrate buffer solution (from kit). Lyophilized ATP standard solution is reconstituted in deionized water to give a 10 mM stock. For the five control wells, 10 μL of serially diluted PACKARD standard is added to each of the standard curve control wells to yield a final concentration in each subsequent well of 200 nM, 100 nM, 50 iiM. 25 nM, and 12.5 nM. PACKARD substrate solution (50 μL) is added to all wells, which are then covered, and the plates are shaken at approximately 700 rpm on a suitable shaker for 2 min. A white PACKARD sticker is attached to the bottom of each plate and samples are dark adapted by wrapping plates in foil and placing in the dark for 10 min. Luminescence is then measured at 22°C using a luminescence counter (e.g., PACKARD TOPCOLTNT Microplate Scintillation and Luminescence Counter or TECAN SPECTRAFLUOR PLUS), and ATP levels calculated from the standard curve. ATP levels in cells treated with test compound(s) are compared to the levels determined for untreated cells. Cells treated with 10 μM of a preferred test compound exhibit ATP levels that are at least 80%, preferably at least 90%, of the untreated cells. When a 100 μM concentration of the test compound is used, cells treated with preferred test compounds exhibit ATP levels that are at least 50%, preferably at least 80%). of the ATP levels detected in untreated cells.

Claims

CLAIMSWhat is claimed is:
1. A compound of the formula:
^44
Figure imgf000074_0001
or a pharmaceutically acceptable salt or hydrate thereof, wherein: A, B, D and E are each CH or N, such that no more than one of A, B, D and E is N; Y is a group of the formula (CH2)r-Q-(CH2)p, which is substituted with from 0 to 4 substituents independently chosen from (i) amino, hydroxy, cyano. C1-C6alkyi, CvCealkenyl, C2-C<;alkynyl and C1-C6haloalkyϊ; and (ii) substituents of the same carbon atom or adjacent carbon atoms that are taken together to form QrQcycIoalkyf, wherein:
Q is absent, CHR10, CH=CH, O, S or NR,0; wherein R10 is hydrogen, C1-Qalkyl or taken together with R3 to form a 4- to 7-membered heterocycloalkyl; and r and p are independently chosen integers ranging from 0 to 6, such that the sum of r and p ranges from 1 to 6;
Rj represents from 0 to 4 substituents independently chosen from halogen, hydroxy, cyano, amino, nitro, aminocarbonyl, aminosulfonyl, -COOH, C]-C6alkyl, C2-C6aikenyl, C2-C6alkynyl, (C3- Cscycioalkyl)C0-C4alkyl, CrC6alkoxy, C1-C6alkylthio, C1-C6alkylsu]finyl, C1-Cgalkoxycarbonyl, C1-CόalkylsulfonylCo-C4alkyl, and mono- or di-(C1-C6aIkyl)aminoC0-C4alkyl;
R2 is C1-C6alkyl, C2-C6aikenyl, C2-C6alkynyl, (C3-C3cycloalkyl)Co-C4alkyl, mono- or di-(C1- C6alky!)aminoC]-C,jalkyl, (4- to 7-membered heterocyc!oalkyl)Co-C4alkyl, pheny!Co-C4alkyl or (5- or 6-membered heteroaryl)Co-C4aikyl, each of which is substituted with from 0 to 4 substituents independently chosen from amino, halogen, hydroxy, cyano. oxo, CpQalkyl, (C3- C8cycloalkyl)Co-C4alkyl and C]-C6alkoxy; R3 is hydrogen, C1-Cealkyl or taken together with Ri0 to form a 4- to 7-membered heterocycloalkyl: and R4 and R5 are:
(i) independently chosen from C]-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, (C3-Qcyclσalky I)Co- Gjalkyl, (4- to 7-membered heterocycloalkyS)Cci-C4aikyI, phenylC0-C4alkyl and (5- to 10- membered heteroaryl)C0-C4aIkyl; or
(ii) taken together to form a 4- to 7-membered heterocycloaikyl; each of which (i) and (ii) is substituted with from 0 to 6 substituents independently chosen from: (a) halogen, hydroxy, cyano, amino, nitro, -COOH, aminocarbonyl and oxo; and (b) C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, (C3-C3cycloalkyl)C0-C4alkyl. mono- or di-(C]-C6alkyl)amino, (4- to 7-membered heterocycloalky])Co-C4alkyϊ, pheny!Co-C4alkyI, (5- or 6-membered heteroaryl)Co-C4alkyl, and substituents that are taken together to form a fused or spiro 5- to 7-membered ring; each of which (b) is substituted with from 0 to 4 substituents independently chosen from:
(1) amino, halogen, hydroxy, cyano, -COOH, aminocarbonyl and oxo; and
(2) C1-C6alkyl, C1-C6aikoxy, C2-C6alkyl ether, mono- or di-(C1-C6a]kyJ)aminoC0-C4alkyl. (C3-C8cycloalkyl)Co-C4alkyl, (4- to 7-membered heterocycloalkyl)Co-C4alkyl, phenylCo-C4alkyl and (5- or 6-mcmbered heteroaiyl)C0-C4alkyl, each of which is substituted with from 0 to 4 substituents independently chosen from halogen, oxo and C1-C4alkyl.
2. A compound or salt or hydrate thereof according to claim 1, wherein R1 represents from 0 to 2 substituents independently chosen from halogen, cyano, and C1-C4aIkyl.
3. A compound or salt or hydrate thereof according to claim 1 or claim 2, wherein R2 is phenylC1-C2aikyl that is substituted with from 0 to 3 substituents independently chosen from halogen, hydroxy, cyano, C]-C4alkyl, and C1-C4aIkoxy.
4. A compound or salt or hydrate thereof according to claim 3. wherein R2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, C]-C4alkyl, and C1-C4aikoxy.
5. A compound or salt or hydrate thereof according to claim 1 or claim 2, wherein R2 is (4- to 7-membered heterocycloalkyl)C1-C4alkyl that is substituted with from 0 to 3 substituents independently chosen from oxo and C1-C4alkyl.
6. A compound or sait or hydrate thereof according to any one of claims 1 -5. wherein Y is CrQalkyiene that is nnsnbstimted or substituted wiih amino, hydroxy, cyano. C1-C<a!kyl, C2- C4alkenyl or C1-C4haloalkyl.
7. A compound or salt or hydrate thereof according to claim 6, wherein Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
8. A compound or salt or hydrate thereof according to any one of claims 1-5, wherein Y is (CHi)1 -CHR lo-, and wherein R10 is taken together with R, to form a 4- to 7-mεmbered heterocycloalkyl.
9. A compound or salt or hydrate thereof according to claim 8, wherein the compound has the formula:
Figure imgf000076_0001
wherein m is 0, 1 , 2 or 3.
10. A compound or salt or hydrate thereof according to any one of claims 1-7, wherein R3 is hydrogen or C1-C4alkyl.
1 1. A compound or salt or hydrate thereof according to any one of claims 1 -10, wherein R4 and R5 are taken together to form a 4- to 7-membered heterocycloalkyl that is substituted with mono- or di-(C1 -C6alkyl)aminoCo-C4alkyl, (4- to 7-membered heterocycloaIkyl)C0-C4alky!, phenylQr C4alkyl or (6-membered heteroaryl)C0-C4alkyl, each of which is unsubstituted or substituted with oxo, C]-C4alkyl or (5- or 6-membered helerocycle)Co-C2alkyl.
12. A compound or salt or hydrate thereof according to claim 1 1 , wherein the compound satisfies the formula:
Figure imgf000076_0002
wherein:
Z is N or CH; and
Rc is piperidinylCo-Gialkyl, pyrrolidinylCo-^alkyl, pyridinylC0-C4alkyl, pyrimidinyIC0-C4aIkyl, trtrahydropyrimidinylCo-C4alkyl, imidazolylCo-Qalkyl. dihydroimidazolylQrQalkyl, thiazolylCo-C4alkyi, oxadiazolylCo-C4aIkyl, phenylCo-C4alkyl, or di-(C!-C4alkyl)aminoCo- Cialkvl, each of which is unsubstituted or substituted with one or two Ci-dalkvl.
13. A compound or salt or hydrate thereof according to claim 1 1 or claim 12, wherein: R] represents from 0 to 2 sυbstituents independently chosen from halogen, cyano, and C1-C4alkyl;
R2 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, C1
C4 alky I, and C]-C4alkoxy; R^ is hydrogen or C1-C4alkyl; and Y is methylene or ethylene, each of which is unsubstituted or substituted with methy! or ethyl.
14. A compound or salt or hydrate thereof according to any one of claims 1-10, wherein: R4 is C1-C4alkyl; and R5 is phenylCo-C2alkyl that is substituted with mono- or di-(C,-C6alkyi)aminoCo-C4alkyl, (4~ to 7- membered heterocycloaIkyl)Co-C4alkyi, phenylCo-C4alkyl or (6-membered heteroaryl)C0-C4alkyl, each of which is unsubstituted or substituted with a oxo, C1-C4alkyl, (5- or 6-membered heleiOcycle)Co-C2alkyl, or mono- or di-(C]-C6alkyl)aminoCo~C4alkyl.
15. A compound or salt or hydrate thereof according to claim 14, wherein the compound satisfies the formula:
Figure imgf000077_0001
wherein:
R7 is methyl or ethyl; and
Rg is piperidinylC0-C4alkyl, pyrrolidinylCo-Qalkyl, pyridinylCo-C4alkyl, pyrimidinylCo-C4alkyl, trtrahydropyrimidinylCo-C4alkyl, imidazolylCo^alkyl. dihydroimidazolyICo-C4alkyl, thiazolyϊCo-C4aIkyI, oxadiazoIyICo-C4alkyl, pheny!Co-C4alkyi, or di-(Cι-C4alkyI)aminoCo- Qalkyl, each of which is unsubstituted or substituted with one or two C1-C4alkyl.
16. A compound or salt or hydrate thereof according to claim 14 or claim 15. wherein: R] represents from 0 to 2 substitucnts independently chosen from halogen, cyano, and Q^alkyi;
R2 is benzyi that is substituted with from 0 to 3 substituents independently chosen from halogen, C1-
C4alkyl, and C1-C4aikoxy; Ri is hydrogen or C,-C4alkyl; and Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
17. A compound or salt or hydrate thereof according to any one of claims 1 -10, wherein R1 and R5 are taken together to form a 8- to 14-membered spiro heterocycloalkyi that is unsubstituted or substituted with C1-C4alkyl. (C3-C7cycloaIkyl)Co-C2alkyI or (5- or 6-membered heierocycle)C0- C2alkyl.
18. A compound or salt or hydrate thereof according to claim 17, wherein the compound satisfies the formula:
Figure imgf000077_0002
wherein: each n is independently 0 or I ; X is O or NR9; and R9 is C]-C4a!kyl or (C3-C7cyc]oalkyl)Co-C2aIky].
19. A compound or salt or hydrate thereof according to claim 17 or claim 18, wherein: R] represents from 0 to 2 substituents independently chosen from halogen, cyano, and C1-C4alkyl; R3 is benzyl that is substituted with from 0 to 3 substituents independently chosen from halogen, C1-
C4a!kyl, and Q^alkoxy; R3 is hydrogen or C1-C4alkyl; and Y is methylene or ethylene, each of which is unsubstituted or substituted with methyl or ethyl.
20. A compound or salt or hydrate thereof according to any one of claims 1-19, wherein A, B, D and E are each CH.
21. A compound or salt or hydrate thereof according to any one of claims 1 -20, wherein the compound exhibits no detectable agonist activity an in vitro assay Of B1 agonism.
22. A compound or salt or hydrate thereof according to any one of claims 1-21 , wherein the compound has an IC50 value of ] micromolar or less in an in vitro assay of B, antagonism.
23. A pharmaceutical composition, comprising at least one compound or salt or hydrate thereof according to any one of claims 1-22, in combination with a physiologically acceptable carrier or excipient.
24. A pharmaceutical composition according to claim 23, wherein the composition is formulated as an injectibie fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup or a transdermal patch.
25. A method for inhibiting induction of agonist-induced Bs activity in vitro, the method comprising contacting B] receptor with at least one compound or salt or hydrate thereof according to any one of claims 1 -22, under conditions and in an amount sufficient to detectabiy inhibit agonist- induced B] acLiviiy,
26. A method for inhibiting induction of agonist-induced B1 activity in a patient, comprising contacting cells expressing B1 receptor with at least one compound or salt or hydrate thereof according to any one of claims 1-22, in an amount sufficient to detectabiy inhibit agonist- induced B i activity in cells expressing a cloned B\ receptor in vitro, and thereby inhibiting agonist- induced B] activity in the patient.
27. A method according to claim 26, wherein the patient is a human.
28. A method for treating a condition responsive to B] receptor modulation in a patient, comprising administering to the patient a therapeutically effective amount of at least one compound or salt or hydrate thereof according to any one of claims 1-22, and thereby alleviating the condition in the patient.
29. A method according to claim 28, wherein the condition is inflammation or pain.
30. A method according to claim 28, wherein the condition is cough, asthma, vascular edema, or epilepsy.
31. A method for treating pain in a patient, comprising administering to a patient suffering from pain a therapeutically effective amount of at feast one compound or salt or hydrate thereof according to any one of claims 1-22, and thereby alleviating pain in the patient.
32. A method according to claim 31 , wherein the patient is suffering from inflammatory pain, acute pain, dental pain, back pain, surgical pain, headache, neuropathic pain or pain from osteoarthritis or trauma.
33. A method according to claim 31, wherein the patient is a human.
34. A compound or salt or hydrate thereof according to claim 1 , wherein the compound is radiolabeled.
35. A method for determining the presence or absence of B] receptor in a sample, comprising the steps of:
(a) contacting a sample with a compound or salt or hydrate thereof according to any one of claims 1 -22, under conditions that permit binding of the compound to B] receptor; and
(b) detecting a signal indicative of a level of the compound bound to Bj receptor, and therefrom determining the presence or absence of B] receptor in the sample.
36. A method according to claim 35, wherein the compound is radiolabeled, and wherein the step of detection comprises the steps of:
(i) separating unbound compound from bound compound; and
(ii) detecting the presence or absence of bound radiolabel in the sample.
37. A packaged pharmaceutical preparation, comprising:
(a) a pharmaceutical composition according to claim 23 in a container; and
(b) instructions for using the composition to treat pain,
38. The use of a compound or salt or hydrate thereof according to any one of claims 1-22 for the manufacture of a medicament for the treatment of a condition responsive to B; receptor modulation.
39. A use according to claim 37, wherein the condition is an inflammatory condition or pain.
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