WO2003079973A2 - Mitotic kinesin inhibitors - Google Patents

Mitotic kinesin inhibitors Download PDF

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Publication number
WO2003079973A2
WO2003079973A2 PCT/US2003/006403 US0306403W WO03079973A2 WO 2003079973 A2 WO2003079973 A2 WO 2003079973A2 US 0306403 W US0306403 W US 0306403W WO 03079973 A2 WO03079973 A2 WO 03079973A2
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Prior art keywords
difluorophenyl
nme
dihydro
alkyl
phenyl
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PCT/US2003/006403
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French (fr)
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WO2003079973A3 (en
Inventor
Michael J. Breslin
Paul J. Coleman
Christopher D. Cox
J. Christopher Culberson
George D. Hartman
Brenda J. Mariano
Maricel Torrent
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Merck & Co., Inc.
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Priority to AU2003249597A priority Critical patent/AU2003249597B2/en
Priority to CA2478068A priority patent/CA2478068C/en
Priority to EP03745083A priority patent/EP1492487B1/en
Priority to JP2003577806A priority patent/JP4399269B2/en
Priority to AT03745083T priority patent/ATE448207T1/en
Priority to DE60329990T priority patent/DE60329990D1/en
Priority to US10/506,702 priority patent/US7745639B2/en
Publication of WO2003079973A2 publication Critical patent/WO2003079973A2/en
Publication of WO2003079973A3 publication Critical patent/WO2003079973A3/en

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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
    • C07D401/04—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 directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/622—Forming processes; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/626—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B
    • C04B35/63—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B using additives specially adapted for forming the products, e.g.. binder binders
    • C04B35/632—Organic additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/06—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • 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
    • C07D401/02—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
    • 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
    • C07D403/04—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 directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D403/06—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 carbon chain containing only aliphatic carbon atoms
    • 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
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom 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
    • C07D413/04—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 directly linked by a ring-member-to-ring-member bond
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    • 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
    • C07D417/04—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 directly linked by a ring-member-to-ring-member bond

Definitions

  • This invention relates to 4,5-dihydropyrazole derivatives that are inhibitors of mitotic kinesins, in particular the mitotic kinesin KSP, and are useful in the treatment of cellular proliferative diseases, for example cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.
  • Taxanes and vinca alkaloids act on microtubules, which are present in a variety of cellular structures.
  • Microtubules are the primary structural element of the mitotic spindle. The mitotic spindle is responsible for distribution of replicate copies of the genome to each of the two daughter cells that result from cell division. It is presumed that disruption of the mitotic spindle by these drugs results in inhibition of cancer cell division, and induction of cancer cell death.
  • microtubules form other types of cellular structures, including tracks for intracellular transport in nerve processes. Because these agents do not specifically target mitotic spindles, they have side effects that limit their usefulness.
  • Mitotic kinesins are enzymes essential for assembly and function of the mitotic spindle, but are not generally part of other microtubule structures, such as in nerve processes. Mitotic kinesins play essential roles during all phases of mitosis. These enzymes are "molecular motors" that transform energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules. The catalytic domain sufficient for this task is a compact structure of approximately 340 amino acids. During mitosis, kinesins organize microtubules into the bipolar structure that is the mitotic spindle.
  • Kinesins mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis.
  • Experimental perturbation of mitotic kinesin function causes malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest and cell death.
  • KSP belongs to an evolutionarily conserved kinesin subfamily of plus end-directed microtubule motors that assemble into bipolar homotetramers consisting of antiparallel homodimers.
  • KSP associates with microtubules of the mitotic spindle.
  • KSP antibodies directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest and induction of programmed cell death.
  • KSP and related kinesins in other, non-human, organisms bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart.
  • KSP may also mediate in anaphase B spindle elongation and focussing of microtubules at the spindle pole.
  • HsEg5 Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42 (1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J.
  • Mitotic kinesins are attractive targets for the discovery and development of novel mitotic chemotherapeutics. Accordingly, it is an object of the present invention to provide compounds, methods and compositions useful in the inhibition of KSP, a mitotic kinesin.
  • the present invention relates to 4,5-dihydropyrazole derivatives, that are useful for treating cellular proliferative diseases, for treating disorders associated with KSP kinesin activity, and for inhibiting KSP kinesin.
  • the compounds of the invention may be illustrated by the Formula I:
  • Rl is selected from:
  • R2 is selected from: 1) C1-C10 alkyl,
  • heterocyclyl said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
  • R3, R4, R5 and R6 are independently selected from:
  • heterocyclyl said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
  • R3 and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(O) m , -N(R9)C(O)-, and -N(COR 10 )-;
  • R9 and RlO are independently selected from:
  • R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
  • R a is (C ⁇ -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl;
  • X is selected from O and S;
  • Rl is selected from:
  • R2 is selected from:
  • heterocyclyl said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
  • R3, R , R5 an d R6 are independently selected from:
  • heterocyclyl said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
  • R and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(O) m , -N(R9)C(O)-, and -N(COR 10 )-;
  • R8 is selected from:
  • R9 and RlO a re independently selected from:
  • R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
  • R a is (C -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl;
  • X is selected from O and S;
  • a further embodiment of the present invention is illustrated by a compound of Formula II, or a pharmaceutically acceptable salt or stereoisomer;
  • Rl is selected from:
  • R2 is selected from: 1) Cl-CiO alkyl,
  • heterocyclyl said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
  • R3, R4, R5 an d R6 are independently selected from:
  • heterocyclyl said alkyl, ; aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
  • R7 is:
  • R9 and RlO are independently selected from:
  • R9 and R O can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one, two or three substituents selected from R8;
  • R a is (C ⁇ -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl;
  • Rl is selected from:
  • R2 is selected from:
  • heteroaryl said alkyl, aryl and heteroaryl is optionally substituted with one or more substituents selected from R7;
  • R3 and R4 are independently selected from:
  • R5 and R6 are independently selected from: 1) H,
  • heterocyclyl said alkyl, aryl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
  • R7, R8 ; R9 ; R10 ; Ra and Rb are as described immediately above.
  • one of R5 and R6 are selected from phenyl or pyridyl, optionally substituted with one or two substituents selected from R7.
  • Another embodiment is the compound of the Formula II described immediately above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is phenyl, optionally substituted with one or two substituents selected from R7.
  • the compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention.
  • the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
  • any variable e.g. R7, R8, R9 5 e t .
  • its definition on each occurrence is independent at every other occurrence.
  • combinations of substituents and variables are permissible only if such combinations result in stable compounds.
  • Lines drawn into the ring systems from substituents represent that the indicated bond may be attached to any of the substitutable ring atoms. If the ring system is polycyclic, it is intended that the bond be attached to any of the suitable carbon atoms on the proximal ring only.
  • substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
  • the phrase "optionally substituted with one or more substituents” should be taken to be equivalent to the phrase “optionally substituted with at least one substituent” and in such cases the preferred embodiment will have from zero to three substituents.
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
  • Ci-Cio as in “Ci-Cio alkyl” is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement.
  • “Ci-Cio alkyl” specifically includes methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, /-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.
  • cycloalkyl means a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms.
  • cycloalkyl includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2- ethyl-cyclopentyl, cyclohexyl, and so on.
  • C1-C6 aralkyl and “C1-C6 heteroaralkyl” the term “C1-C6” refers to the alkyl portion of the moiety and does not describe the number of atoms in the aryl and heteroaryl portion of the moiety.
  • Alkoxy represents either a cyclic or non-cyclic alkyl group of indicated number of carbon atoms attached through an oxygen bridge. "Alkoxy” therefore encompasses the definitions of alkyl and cycloalkyl above. If no number of carbon atoms is specified, the term “alkenyl” refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl” means an alkenyl radical having from 2 to 6 carbon atoms.
  • Alkenyl groups include ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl.
  • the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.
  • alkynyl refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present.
  • C2-C6 alkynyl means an alkynyl radical having from 2 to 6 carbon atoms.
  • Alkynyl groups include ethynyl, propynyl, butynyl, 3-methylbutynyl and so on.
  • the straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
  • substituents may be defined with a range of carbons that includes zero, such as (Co-C6)alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as -CH2Ph, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph, and so on.
  • aryl is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic.
  • aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl.
  • the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
  • heteroaryl represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline.
  • heteroaryl is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl.
  • heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively.
  • heterocycle or “heterocyclyl” as used herein is intended to mean a 3- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • Heterocyclyl therefore includes the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof. Further examples of “heterocyclyl” include, but are not limited to the following: azetindinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl
  • heterocycle or “heterocyclyl” as used herein is intended to mean a 5- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups.
  • Heterocyclyl therefore includes, in this embodiment, the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof.
  • heterocyclyl include, but are not limited to the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridazinyl
  • heterocycle is selected from 2-azepinone, benzimidazolyl, 2-diazapinone, imidazolyl, 2-imidazolidinone, indolyl, isoquinolinyl, mo ⁇ holinyl, piperidyl, piperazinyl, pyridyl, pyrrolidinyl, 2-piperidinone, 2-pyrimidinone, 2- pyrollidinone, quinolinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, and thienyl.
  • halo or halogen as used herein is intended to include chloro, fluoro, bromo and iodo.
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • a (C ⁇ -C6)alkyl may be substituted with one, two or three substituents selected from OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as mo ⁇ holinyl, piperidinyl, and so on.
  • cyclic moieties may optionally include a heteroatom(s).
  • heteroatom-containing cyclic moieties include, but are not limited to:
  • R9 and RlO are defined such that they can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said heterocycle optionally substituted with one or more substituents selected from R8.
  • heterocycles that can thus be formed include, but are not limited to the following, keeping in mind that the heterocycle is optionally substituted with one or more (and preferably one, two or three) substituents chosen from R ;
  • R is selected from aryl, optionally substituted with one to three substituents selected from R7. More preferably, R2 is phenyl, optionally substituted with one to three substituents selected from halo.
  • R5 and R6 are selected from H, C1-C6 alkyl, aryl and heteroaryl, optionally substituted with one to two substituents selected from R7. More preferably, one of R5 and R6 is phenyl, optionally substituted with one substituents selected from R7, and the other of R5 and R6 is H or C1-C6 alkyl. Still more preferably, one of R5 and R6 is phenyl, optionally 3-substituted with one substituents selected from OH and NH2, and the other of R5 and R6 is H or C1-C6 alkyl.
  • the free form of compounds of Formula I is the free form of compounds of Formula I, as well as the pharmaceutically acceptable salts and stereoisomers thereof.
  • Some of the specific compounds exemplified herein are the protonated salts of amine compounds.
  • the term "free form” refers to the amine compounds in non-salt form.
  • the encompassed pharmaceutically acceptable salts not only include the salts exemplified for the specific compounds described herein, but also all the typical pharmaceutically acceptable salts of the free form of compounds of Formula I.
  • the free form of the specific salt compounds described may be isolated using techniques known in the art.
  • the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate.
  • the free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for pu ⁇ oses of the invention.
  • the pharmaceutically acceptable salts of the instant compounds can be synthesized from the compounds of this invention which contain a basic or acidic moiety by conventional chemical methods.
  • the salts of the basic compounds are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
  • the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base.
  • pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed by reacting a basic instant compound with an inorganic or organic acid.
  • non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy- benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like
  • organic acids such as acetic, propionic, succinic, glycolic, ste
  • suitable “pharmaceutically acceptable salts” refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases.
  • Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, N-N'-dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmo ⁇ holine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, mo ⁇ holine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.
  • basic ion exchange resins such as arginine
  • the compounds of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom.
  • the compounds of this invention may be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature or exemplified in the experimental procedures. The illustrative schemes below, therefore, are not limited by the compounds listed or by any particular substituents employed for illustrative pu ⁇ oses. Substituent numbering as shown in the schemes does not necessarily correlate to that used in the claims and often, for clarity, a single substituent is shown attached to the compound where multiple substituents are allowed under the definitions of Formula I hereinabove.
  • condensation of a suitably substituted acetophenone A-l with a suitably substituted benzaldehyde A-2 provides the ⁇ - hydroxylcarbonyl intermediate A-3.
  • Dehydration of A-3 with trifluoroacetic anhydride provides the ⁇ , ⁇ -unsaturated carbonyl compound A-4.
  • Intermediate A-4 can then undergo reaction with hydrazine in the presence of a carboxylic acid A-5 to provide the N-acyldihydropyrazole A-6.
  • reaction of the intermediate A-4 with hydrazine may be performed without the carboyxlic acid and the intermediate can then be reacted with a variety of acetylating and electrophilic reagents.
  • Scheme B also illustrates the preparation of the instant compounds by reaction of the A-4 intermediate with an N-substituted hydrazine to provide the instant compound B-3.
  • Scheme C illustrates preparation of the 5,5-disubstituted compound of the instant invention.
  • reaction of the intermediate A-4 with, for example, a cuprate reagent provides intermediate C-l, which can then be dehydrated to provide the substituted chalcone C-2.
  • Subsequent reaction with a hydrazine, as shown provides the compound of the instant invention.
  • reaction sequence shown in scheme A is generally applicable to preparing other compounds of the instant invention that have
  • R and R /R other than phenyl.
  • Scheme E illustrates preparation of the 5,5-disubstituted compound having a functional moiety (a hydroxy) which can then be further functionalized.
  • Scheme F shows a synthetic method for preparing compounds of the instant invention having 1,3,4,5-tetrasubstitution.
  • the synthetic route is analogous to the method described in Scheme A but starting with a substituted acetophenone F-l.
  • mitosis may be altered in a variety of ways; that is, one can affect mitosis either by increasing or decreasing the activity of a component in the mitotic pathway. Stated differently, mitosis may be affected (e.g., disrupted) by disturbing equilibrium, either by inhibiting or activating certain components. Similar approaches may be used to alter meiosis.
  • the compounds of the invention are used to modulate mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis.
  • modulate herein is meant altering mitotic spindle formation, including increasing and decreasing spindle formation.
  • mitotic spindle formation herein is meant organization of microtubules into bipolar structures by mitotic kinesins.
  • mitotic spindle dysfunction herein is meant mitotic arrest and monopolar spindle formation.
  • the compounds of the invention are useful to bind to and/or modulate the activity of a mitotic kinesin.
  • the mitotic kinesin is a member of the bimC subfamily of mitotic kinesins (as described in U.S. Patent No. 6,284,480, column 5).
  • the mitotic kinesin is human KSP, although the activity of mitotic kinesins from other organisms may also be modulated by the compounds of the present invention.
  • modulate means either increasing or decreasing spindle pole separation, causing malformation, i.e., splaying, of mitotic spindle poles, or otherwise causing mo ⁇ hological perturbation of the mitotic spindle.
  • KSP KSP for these pu ⁇ oses
  • variants and/or fragments of KSP See PCT Publ. WO 01/31335: "Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell Proliferation States", filed Oct. 27, 1999, hereby inco ⁇ orated by reference in its entirety.
  • other mitotic kinesins may be inhibited by the compounds of the present invention.
  • the compounds of the invention are used to treat cellular proliferation diseases.
  • Disease states which can be treated by the methods and compositions provided herein include, but are not limited to, cancer (further discussed below), autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. It is appreciated that in some cases the cells may not be in a hyper- or hypoproliferation state (abnormal state) and still require treatment. For example, during wound healing, the cells may be proliferating "normally", but proliferation enhancement may be desired.
  • cells may be in a "normal" state, but proliferation modulation may be desired to enhance a crop by directly enhancing growth of a crop, or by inhibiting the growth of a plant or organism which adversely affects the crop.
  • the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders or states.
  • cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, a
  • the term "cancerous cell” as provided herein includes a cell afflicted by any one of the above-identified conditions.
  • the compounds of the instant invention may also be useful as antifungal agents, by modulating the activity of the fungal members of the bimC kinesin subgroup, as is described in U.S. Patent No. 6,284,480.
  • the compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
  • the compounds of the instant invention may be administered to a mammal in need thereof using a gel extrusion mechanism (GEM) device, such as that described in USSN 60/144,643, filed on July 20, 1999, which is hereby inco ⁇ orated by reference.
  • GEM gel extrusion mechanism
  • composition is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
  • the pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and abso ⁇ tion in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a water soluble taste masking material such as hydroxypropyl-methylcellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate 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, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene- oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
  • 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, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • the pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening, flavoring agents, preservatives and antioxidants.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may also be a sterile injectable oil-in- water microemulsion where the active ingredient is dissolved in the oily phase.
  • the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
  • the injectable solutions or microemulsions may be introduced into a patient's blood stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound.
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this pu ⁇ ose any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • creams, ointments, jellies, solutions or suspensions, etc. containing the compound of Formula I are employed. (For pu ⁇ oses of this application, topical application shall include mouth washes and gargles.)
  • the compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.
  • a suitable amount of compound is administered to a mammal undergoing treatment for cancer.
  • Administration occurs in an amount between about 0.1 mg kg of body weight to about 60 mg kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per day.
  • the instant compounds are also useful in combination with known therapeutic agents and anti-cancer agents.
  • the instant compounds are useful in combination with known anti-cancer agents.
  • Combinations of the presently disclosed compounds with other anti-cancer or chemotherapeutic agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6 th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers.
  • a person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved.
  • anti-cancer agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, and agents that interfere with cell cycle checkpoints.
  • the instant compounds are particularly useful when co-administered with radiation therapy.
  • the instant compounds are also useful in combination with known anti-cancer agents including the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl -protein transferase inhibitors, HMG- CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, and other angiogenesis inhibitors.
  • Estrogen receptor modulators refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism.
  • estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2- dimethyl- 1 -oxopropoxy-4-methyl-2- [4- [2-( 1 -piperidinyl)ethoxy] phenyl] -2H- 1 - benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4'-dihydroxybenzophenone-2,4- dinitrophenyl-hydrazone, and SH646.
  • Androgen receptor modulators refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism.
  • Examples of androgen receptor modulators include finasteride and other 5 ⁇ -reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
  • Retinoid receptor modulators refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism.
  • retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, -difluoromethylornithine, ILX23-7553, trans-N-(4'- hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.
  • Cytotoxic/cytostatic agents refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, antimetabolites; biological response modifiers; hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors and ubiquitin ligase inhibitors.
  • cytotoxic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-l,6- diamine)-m
  • proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).
  • microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'- norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, obulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6- pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L- proline-t-butylamide, TDX258, the epothilones (see for example U.S.
  • epothilones are not included in the microtubule inhibitors/microtubule-stabilising agents.
  • topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzylidene- chartreusin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH, 12H- benzo[de]pyrano[3' ,4' :b,7]-indolizino[l ,2b]quinoline-10, 13(9H, 15H)dione, lurtotecan
  • inhibitors of mitotic kinesins are described in PCT Publications WO 01/30768, WO 01/98278 and WO 02/078639, and pending U.S. Ser. Nos. 60/338,779 (filed December 6, 2001), 60/338,344 (filed December 6, 2001), 60/338,383 (filed December 6, 2001), 60/338,380 (filed December 6, 2001), 60/338,379 (filed December 6, 2001) and 60/344,453 (filed November 7, 2001).
  • inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK, inhibitors of aurora kinase and inhibitors of Rab6-KJFL.
  • Antiproliferative agents includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'-fluoromethylene-2'- deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)ure
  • Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.
  • HMG-CoA reductase inhibitors refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. Compounds which have inhibitory activity for HMG- CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Patent 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33.
  • the terms "HMG-CoA reductase inhibitor” and “inhibitor of HMG-CoA reductase” have the same meaning when used herein.
  • HMG-CoA reductase inhibitors examples include but are not limited to lovastatin (MEVACOR®; see U.S. Patent Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Patent Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Patent Nos.
  • HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.
  • An illustration of the lactone portion and its corresponding open-acid form is shown below as structures I and ⁇ .
  • HMG-CoA reductase inhibitors where an open-acid form can exist
  • salt and ester forms may be formed from the open-acid, and all such forms are included within the meaning of the term "HMG-CoA reductase inhibitor" as used herein.
  • the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and in a further embodiment, simvastatin.
  • the term "pharmaceutically acceptable salts" with respect to the HMG-CoA reductase inhibitor shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base, particularly those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium, as well as those salts formed from amines such as ammonia, ethylenediamine, N- methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, 1-p- chlorobenzyl-2-pyrrolidine-l'-yl-methylbenz-imidazole, diethylamine, piperazine, and tris(hydroxymethyl) aminomethane.
  • a suitable organic or inorganic base particularly those formed from cations such
  • salt forms of HMG- CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamao
  • Ester derivatives of the described HMG-CoA reductase inhibitor compounds may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.
  • Prenyl-protein transferase inhibitor refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-13 (GGPTase-JJ, also called Rab GGPTase).
  • prenyl-protein transferase inhibiting compounds examples include (+)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-5-yl)methyl]-4-(3- chlorophenyl)- 1 -methyl-2( lH)-quinolinone, (-)-6-[amino(4-chlorophenyl)( 1 -methyl - l ⁇ -imidazol-5-yl)methyl]-4-(3-chlorophenyl)-l-methyl-2(lH)-quinolinone, (+)-6- [amino(4-chlorophenyl)(l-methyl-l ⁇ -imidazol-5-yl) methyl]-4-(3-chlorophenyl)-l- methyl-2(lH)-quinolinone, 5(S)-n-butyl-l-(2,3-dimethylphenyl)-4-[l-(4- cyanobenzyl)-5
  • prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ.
  • Angiogenesis inhibitors refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.
  • angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon- ⁇ , interleukin- 12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti- inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy- genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol.
  • NSAIDs nonsteroidal anti- inflammatories
  • NSAIDs nonsteroidal anti
  • steroidal anti-inflammatories such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)- fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med.
  • agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)).
  • agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80: 10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)).
  • TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed August 8, 2001) and 60/349,925 (filed January 18, 2002).
  • Agents that interfere with cell cycle checkpoints refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents.
  • agents include inhibitors of ATR, ATM, the Chkl and Chk2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.
  • “Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors.
  • Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138), inhibitors of Raf kinase (for example BAY-43- 9006 ), inhibitors of MEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).
  • inhibitors of serine/threonine kinases including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138
  • inhibitors of Raf kinase for example BAY-43- 9006
  • inhibitors of MEK for example CI-1040 and PD-098059
  • inhibitors of mTOR for example Wyeth
  • NSAID's which are potent COX-2 inhibiting agents.
  • an NSAJD is potent if it possess an IC 50 for the inhibition of COX-2 of l ⁇ M or less as measured by cell or microsomal assays.
  • NSAID's which are selective COX-2 inhibitors are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays.
  • Such compounds include, but are not limited to those disclosed in U.S. Patent 5,474,995, issued December 12, 1995, U.S. Patent 5,861,419, issued January 19, 1999, U.S. Patent 6,001,843, issued December 14, 1999, U.S. Patent 6,020,343, issued February 1, 2000, U.S.
  • Patent 5,409,944 issued April 25, 1995, U.S. Patent 5,436,265, issued July 25, 1995, U.S. Patent 5,536,752, issued July 16, 1996, U.S. Patent 5,550,142, issued August 27, 1996, U.S. Patent 5,604,260, issued February 18, 1997, U.S. 5,698,584, issued December 16, 1997, U.S. Patent 5,710,140, issued January 20,1998, WO 94/15932, published July 21, 1994, U.S. Patent 5,344,991, issued June 6, 1994, U.S. Patent 5,134,142, issued July 28, 1992, U.S. Patent 5,380,738, issued January 10, 1995, U.S. Patent 5,393,790, issued February 20, 1995, U.S. Patent 5,466,823, issued November 14, 1995, U.S. Patent 5,633,272, issued May 27, 1997, and U.S. Patent 5,932,598, issued August 3, 1999, all of which are hereby inco ⁇ orated by reference.
  • Inhibitors of COX-2 that are particularly useful in the instant method of treatment are:
  • angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2- butenyl)oxiranyl]-l-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-lH-l,2,3-triazole-4- carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2- pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(l,
  • integrated circuit blockers refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the 0 v ⁇ 3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ v ⁇ 5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the ⁇ v ⁇ 3 integrin and the CCv ⁇ 5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells.
  • the term also refers to antagonists of the ⁇ v ⁇ 6, « v ⁇ 8> oci ⁇ l, ⁇ 2 ⁇ l, ccs ⁇ l, ⁇ 6 ⁇ l and 0C6 ⁇ 4 integrins.
  • the term also refers to antagonists of any combination of ⁇ v ⁇ 3, ⁇ v ⁇ 5, ⁇ ⁇ 6 ' av P 8 > a l ⁇ l> a 2 ⁇ l> «5 ⁇ l> « ⁇ l and ⁇ 4 integrins.
  • tyrosine kinase inhibitors include N- (trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5- yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3- chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-mo ⁇ holinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11, 12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-lH- diindolo[l,2,3-fg:3',2',l'-kl]pyrrolo[3,4-i
  • Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods.
  • combinations of the instantly claimed compounds with PPAR- ⁇ (i.e., PPAR-garnma) agonists and PPAR- ⁇ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies.
  • PPAR- ⁇ and PPAR- ⁇ are the nuclear peroxisome proliferator-activated receptors ⁇ and ⁇ .
  • the expression of PPAR- ⁇ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol Chem. 1999;274:9116-9121; Invest.
  • PPAR- ⁇ agonists and PPAR- ⁇ / ⁇ agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR- H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3- trifluoromethyl-l,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phen
  • Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer.
  • Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Patent No.
  • a uPA/uPAR antagonist (Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998;5(8): 1105-13), and interferon gamma (/ Immunol 2000;164:217-222).
  • the compounds of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins.
  • MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).
  • a compound of the present invention may be employed in conjunction with anti-emetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy.
  • a compound of the present invention may be used in conjunction with other anti- emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos.
  • neurokinin-1 receptor antagonists especially 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos
  • an antidopaminergic such as the phenothiazines (for example prochlo ⁇ erazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol.
  • phenothiazines for example prochlo ⁇ erazine, fluphenazine, thioridazine and mesoridazine
  • metoclopramide metoclopramide or dronabinol.
  • conjunctive therapy with an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is preferred.
  • Neurokinin-1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos.
  • the neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(l- (R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo- lH,4H-l,2,4-triazolo)methyl)mo ⁇ holine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719,147.
  • a compound of the instant invention may also be administered with an agent useful in the treatment of anemia.
  • an anemia treatment agent is, for example, a continuous eythropoiesis receptor activator (such as epoetin alfa).
  • a compound of the instant invention may also be administered with an agent useful in the treatment of neutropenia.
  • a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G- CSF).
  • G- CSF human granulocyte colony stimulating factor
  • Examples of a G-CSF include filgrastim.
  • a compound of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.
  • an immunologic-enhancing drug such as levamisole, isoprinosine and Zadaxin.
  • the scope of the instant invention encompasses the use of the instantly claimed compounds in combination with a second compound selected from: 1) an estrogen receptor modulator,
  • angiogenesis inhibitor 11) an angiogenesis inhibitor, 11) a PPAR- ⁇ agonists,
  • the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal- derived growth factor, an inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metal loprotease) inhibitor, an integrin blocker, interferon- ⁇ , interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl- carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF.
  • the estrogen receptor modulator is tamoxifen or raloxifene.
  • a method of treating cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with radiation therapy and/or in combination with a compound selected from:
  • Yet another embodiment of the invention is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with paclitaxel or trastuzumab.
  • the invention further encompasses a method of treating or preventing cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with a COX-2 inhibitor.
  • the instant invention also includes a pharmaceutical composition useful for treating or preventing cancer that comprises a therapeutically effective amount of a compound of Formula I and a compound selected from:
  • the invention further comprises the use of the instant compounds in a method to screen for other compounds that bind to KSP.
  • the KSP is bound to a support, and a compound of the invention (which is a mitotic agent) is added to the assay.
  • the compound of the invention is bound to the support and KSP is added.
  • Classes of compounds among which novel binding agents may be sought include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for candidate agents that have a low toxicity for human cells.
  • assays may be used for this pu ⁇ ose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
  • the determination of the binding of the mitotic agent to KSP may be done in a number of ways.
  • the mitotic agent (the compound of the invention) is labeled, for example, with a fluorescent or radioactive moiety and binding determined directly.
  • this may be done by attaching all or a portion of KSP to a solid support, adding a labeled mitotic agent (for example a compound of the invention in which at least one atom has been replaced by a detectable isotope), washing off excess reagent, and determining whether the amount of the label is that present on the solid support.
  • a labeled mitotic agent for example a compound of the invention in which at least one atom has been replaced by a detectable isotope
  • washing off excess reagent for example a compound of the invention in which at least one atom has been replaced by a detectable isotope
  • Various blocking and washing steps may be utilized as is known in the art.
  • labeled herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal. In some embodiments, only one of the components is labeled. For example, radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known
  • the kinesin proteins may be labeled at tyrosine positions using I, or with fluorophores.
  • more than one component may be labeled with different labels; using 125 I for the proteins, for example, and a fluorophor for the mitotic agents.
  • the compounds of the invention may also be used as competitors to screen for additional drug candidates.
  • "Candidate bioactive agent” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactivity. They may be capable of directly or indirectly altering the cellular proliferation phenotype or the expression of a cellular proliferation sequence, including both nucleic acid sequences and protein sequences. In other cases, alteration of cellular proliferation protein binding and/or activity is screened. Screens of this sort may be performed either in the presence or absence of microtubules.
  • preferred embodiments exclude molecules already known to bind to that particular protein, for example, polymer structures such as microtubules, and energy sources such as ATP.
  • Preferred embodiments of assays herein include candidate agents which do not bind the cellular proliferation protein in its endogenous native state termed herein as "exogenous" agents.
  • exogenous agents further exclude antibodies to KSP.
  • Candidate agents can encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • Second sample comprises a mitotic agent, KSP and a drug candidate. This may be performed in either the presence or absence of microtubules.
  • the binding of the drug candidate is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to KSP and potentially modulating its activity. That is, if the binding of the drug candidate is different in the second sample relative to the first sample, the drug candidate is capable of binding to KSP.
  • the binding of the candidate agent is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to KSP, such as an antibody, peptide, binding partner, ligand, etc.
  • a binding moiety known to bind to KSP such as an antibody, peptide, binding partner, ligand, etc.
  • the candidate agent is labeled. Either the candidate agent, or the competitor, or both, is added first to KSP for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between about 4 and about 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding. In a preferred embodiment, the competitor is added first, followed by the candidate agent.
  • Displacement of the competitor is an indication the candidate agent is binding to KSP and thus is capable of binding to, and potentially modulating, the activity of KSP.
  • either component can be labeled.
  • the presence of label in the wash solution indicates displacement by the agent.
  • the candidate agent is labeled, the presence of the label on the support indicates displacement.
  • the candidate agent is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor may indicate the candidate agent is bound to KSP with a higher affinity.
  • the candidate agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate the candidate agent is capable of binding to KSP.
  • KSP binding site of KSP. This can be done in a variety of ways. In one embodiment, once KSP has been identified as binding to the mitotic agent, KSP is fragmented or modified and the assays repeated to identify the necessary components for binding.
  • Modulation is tested by screening for candidate agents capable of modulating the activity of KSP comprising the steps of combining a candidate agent with KSP, as above, and determining an alteration in the biological activity of KSP.
  • the candidate agent should both bind to KSP (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods and in vivo screening of cells for alterations in cell cycle distribution, cell viability, or for the presence, mo ⁇ ohology, activity, distribution, or amount of mitotic spindles, as are generally outlined above.
  • differential screening may be used to identify drug candidates that bind to the native KSP, but cannot bind to modified KSP.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non- specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
  • reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • Plasmids for the expression of the human KSP motor domain construct were cloned by PCR using a pBluescript full length human KSP construct (Blangy et al., Cell, vol.83, ppl 159-1169, 1995) as a template.
  • the N-terminal primer 5'- GCAACGATTAATATGGCGTCGCAGCCAAATTCGTCTGCGAAG (SEQ.ID.NO.: 1) and the C-terminal primer 5'-GCAACGCTCGAGTCAGTGAT GATGGTGGTGATGCTGATTCACTTCAGGCTTATTCAATAT (SEQ.ID.NO. : 2) were used to amplify the motor domain and the neck linker region.
  • the PCR products were digested with Asel and Xhol, ligated into the Ndel/Xhol digestion product of pRSETa (Invitrogen) and transformed into E. coli BL21 (DE3).
  • lysis buffer 50mM K-HEPES, pH 8.0, 250mM KC1, 0.1% Tween, lOmM imidazole, 0.5mM Mg-ATP, ImM PMSF, 2mM benzimidine, lx complete protease inhibitor cocktail (Roche)
  • Cell suspensions were incubated with lmg/ml lysozyme and 5mM ⁇ -mercaptoethanol on ice for 10 minutes, followed by sonication (3x 30sec). All subsequent procedures were performed at 4°C. Lysates were centrifuged at 40,000x g for 40 minutes.
  • Supernatants were diluted and loaded onto an SP Sepharose column (Pharmacia, 5ml cartridge) in buffer A (50mM K-HEPES, pH 6.8, ImM MgCl 2 , ImM EGTA, lO ⁇ M Mg-ATP, ImM DTT) and eluted with a 0 to 750mM KC1 gradient in buffer A.
  • buffer A 50mM K-HEPES, pH 6.8, ImM MgCl 2 , ImM EGTA, lO ⁇ M Mg-ATP, ImM DTT
  • Fractions containing KSP were pooled and incubated with Ni-NTA resin (Qiagen) for one hour. The resin was washed three times with buffer B (Lysis buffer minus PMSF and protease inhibitor cocktail), followed by three 15-minute incubations and washes with buffer B.
  • Microtubules are prepared from tubulin isolated from bovine brain. Purified tubulin (> 97% MAP-free) at 1 mg/ml is polymerized at 37°C in the presence of 10 ⁇ M paclitaxel, 1 mM DTT, 1 mM GTP in BRB80 buffer (80 mM K-PIPES, 1 mM EGTA, 1 mM MgCl 2 at pH 6.8). The resulting microtubules are separated from non-polymerized tubulin by ultracentrifugation and removal of the supernatant.
  • the pellet, containing the microtubules, is gently resuspended in 10 ⁇ M paclitaxel, 1 mM DTT, 50 ⁇ g/ml ampicillin, and 5 ⁇ g ml chloramphenicol in BRB80.
  • the kinesin motor domain is incubated with microtubules, 1 mM ATP (1:1 MgCl 2 : Na-ATP), and compound at 23°C in buffer containing 80 mM K-HEPES (pH 7.0), 1 mM EGTA, 1 mM DTT, 1 mM MgCl 2 , and 50 mM KC1.
  • the reaction is terminated by a 2-10 fold dilution with a final buffer composition of 80 mM HEPES and 50 mM EDTA.
  • Free phosphate from the ATP hydrolysis reaction is measured via a quinaldine red/ammonium molybdate assay by adding 150 ⁇ l of quench C buffer containing a 2:1 ratio of quench A:quench B.
  • Quench A contains 0.1 mg/ml quinaldine red and 0.14% polyvinyl alcohol; quench B contains 12.3 mM ammonium molybdate tetrahydrate in 1.15 M sulfuric acid. The reaction is incubated for 10 minutes at 23°C, and the absorbance of the phospho-molybdate complex is measured at 540 nm.
  • Cells are plated in 96-well tissue culture dishes at densities that allow for logarithmic growth over the course of 24, 48, and 72 hours and allowed to adhere overnight. The following day, compounds are added in a 10-point, one-half log titration to all plates. Each titration series is performed in triplicate, and a constant DMSO concentration of 0.1% is maintained throughout the assay. Controls of 0.1% DMSO alone are also included. Each compound dilution series is made in media without serum. The final concentration of serum in the assay is 5% in a 200 ⁇ L volume of media.
  • Alamar blue staining reagent Twenty microliters of Alamar blue staining reagent is added to each sample and control well on the titration plate at 24, 48, or 72 hours following the addition of drug and returned to incubation at 37°C. Alamar blue fluorescence is analyzed 6-12 hours later on a CytoFluor II plate reader using 530-560 nanometer wavelength excitation, 590 nanometer emission.
  • a cytotoxic EC 50 is derived by plotting compound concentration on the x-axis and average percent inhibition of cell growth for each titration point on the y-axis. Growth of cells in control wells that have been treated with vehicle alone is defined as 100% growth for the assay, and the growth of cells treated with compounds is compared to this value. Proprietary in-house software is used to calculate percent cytotoxicity values and inflection points using logistic 4-parameter curve fitting. Percent cytotoxicity is defined as:
  • the inflection point is reported as the cytotoxic EC 50 .
  • FACS FACS analysis is used to evaluate the ability of a compound to arrest cells in mitosis and to induce apoptosis by measuring DNA content in a treated population of cells.
  • Cells are seeded at a density of 1.4x10° cells per 6cm 2 tissue culture dish and allowed to adhere overnight. Cells are then treated with vehicle
  • An EC 50 for mitotic arrest is derived by plotting compound concentration on the x-axis and percentage of cells in the G2/M phase of the cell cycle for each titration point (as measured by propidium iodide fluorescence) on the y-axis. Data analysis is performed using the SigmaPlot program to calculate an inflection point using logistic 4-parameter curve fitting. The inflection point is reported as the EC 50 for mitotic arrest. A similar method is used to determine the compound EC 50 for apoptosis. Here, the percentage of apoptotic cells at each titration point (as determined by propidium iodide fluorescence) is plotted on the y-axis, and a similar analysis is carried out as described above.
  • Paraffin-embedded tumor sections are deparaffinized with xylene and rehydrated through an ethanol series prior to blocking.
  • Slides are incubated in primary antibodies (mouse monoclonal anti- ⁇ -tubulin antibody, clone DM1 A from Sigma diluted 1:500; rabbit polyclonal anti-pericentrin antibody from Covance, diluted 1:2000) overnight at 4°C.
  • primary antibodies mouse monoclonal anti- ⁇ -tubulin antibody, clone DM1 A from Sigma diluted 1:500; rabbit polyclonal anti-pericentrin antibody from Covance, diluted 1:2000
  • conjugated secondary antibodies FITC-conjugated donkey anti- mouse IgG for tubulin; Texas red-conjugated donkey anti-rabbit IgG for pericentrin
  • FITC-conjugated donkey anti- mouse IgG for tubulin Texas red-conjugated donkey anti-rabbit IgG for pericentrin
  • Step 1 3-r3-(benzyloxy)phenyll-l-(2-chlorophenyl)prop-2-en-l-one (1-4)
  • Step 2 l-(2-chlorophenyl)-3-(hvdroxyphenyl)prop-2-en-l-one (1-5) To a solution of 740 mg (2.12mmol) of L in 15 mL of CH 2 C1 2 at
  • this material was dissolved in 2.5 mL of CH 2 C1 , 22 ⁇ L (0.45mmol) of hydrazine hydrate was added, and the mixture was heated at 150°C in a microwave reactor for 5 min. After cooling the reaction to -78°C, 210 mg (1.5mmol) of K 2 CO 3 and 54 ⁇ L (0.75mmol) of acetyl chloride were added, and the reaction was allowed to warm to rt and stir for 12h. The mixture was then dumped into a separatory funnel with 10% aqueous citric acid, extracted twice with CH 2 C1 2 , washed with NaHCO 3 , and dried over Na SO 4 .
  • Step 1 6- ⁇ [tert-butyl(dimethyl)silyl]oxy ⁇ -l-(2,5-difluorophenyl)hex-2-yn-l- one (7-3)
  • Step 3 3-[3-(2,5-difluorophenyl)-l-(mo ⁇ holin-4-ylcarbonyl)-5-phenyl-4,5- dihvdro-lH-pyrazol-5-yllpropan-l-amine (7-7)
  • 1 ⁇ 6 in CH 2 C1 2 was added 175 ⁇ L (1.24 mmol) of triethylamine and 75 ⁇ L (0.93 mmol) of mesyl chloride.
  • the mixture was dumped into a separatory funnel with saturated NaHCO 3 , extracted twice with CH 2 C1 2 , washed with water, and dried over Na 2 SO 4 .
  • a DMF solution was made by mixing 1 mol equivalent of each of the following: amine 10-1, imidazole-2-carboxylic acid, triethylamine, HO AT, and EDCI. The resultant mixture was stirred overnight, and then loaded directly onto a reverse phase HPLC for purification, providing 10 ⁇ as a white solid.
  • R ⁇ R 3 R° R 1 ,5-difluorophenyl H 2-imidazolyl NMe 2 ,5-difluorophenyl H 2- thiazolyl NMe 2 ,5-difluorophenyl H 2-oxazolyl NMe 2 ,5-difluorophenyl JJ 3-isoxazolyl NMe 2

Abstract

The present invention relates to dihydropyrazole compounds that are useful for treating cellular proliferative diseases, for treating disorders associated with KSP kinesin activity, and for inhibiting KSP kinesin. The invention also related to compositions which comprise these compounds, and methods of using them to treat cancer in mammals.

Description

TITLE OF THE INVENTION MITOTIC KINESIN INHIBITORS
BACKGROUND OF THE INVENTION This invention relates to 4,5-dihydropyrazole derivatives that are inhibitors of mitotic kinesins, in particular the mitotic kinesin KSP, and are useful in the treatment of cellular proliferative diseases, for example cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.
Among the therapeutic agents used to treat cancer are the taxanes and vinca alkaloids. Taxanes and vinca alkaloids act on microtubules, which are present in a variety of cellular structures. Microtubules are the primary structural element of the mitotic spindle. The mitotic spindle is responsible for distribution of replicate copies of the genome to each of the two daughter cells that result from cell division. It is presumed that disruption of the mitotic spindle by these drugs results in inhibition of cancer cell division, and induction of cancer cell death. However, microtubules form other types of cellular structures, including tracks for intracellular transport in nerve processes. Because these agents do not specifically target mitotic spindles, they have side effects that limit their usefulness.
Improvements in the specificity of agents used to treat cancer is of considerable interest because of the therapeutic benefits which would be realized if the side effects associated with the administration of these agents could be reduced. Traditionally, dramatic improvements in the treatment of cancer are associated with identification of therapeutic agents acting through novel mechanisms. Examples of this include not only the taxanes, but also the camptothecin class of topoisomerase I inhibitors. From both of these perspectives, mitotic kinesins are attractive targets for new anti-cancer agents.
Mitotic kinesins are enzymes essential for assembly and function of the mitotic spindle, but are not generally part of other microtubule structures, such as in nerve processes. Mitotic kinesins play essential roles during all phases of mitosis. These enzymes are "molecular motors" that transform energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules. The catalytic domain sufficient for this task is a compact structure of approximately 340 amino acids. During mitosis, kinesins organize microtubules into the bipolar structure that is the mitotic spindle. Kinesins mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis. Experimental perturbation of mitotic kinesin function causes malformation or dysfunction of the mitotic spindle, frequently resulting in cell cycle arrest and cell death. Among the mitotic kinesins which have been identified is KSP. KSP belongs to an evolutionarily conserved kinesin subfamily of plus end-directed microtubule motors that assemble into bipolar homotetramers consisting of antiparallel homodimers. During mitosis KSP associates with microtubules of the mitotic spindle. Microinjection of antibodies directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest and induction of programmed cell death. KSP and related kinesins in other, non-human, organisms, bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart. KSP may also mediate in anaphase B spindle elongation and focussing of microtubules at the spindle pole.
Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42 (1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J. Cell Sci., 111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBank accession numbers: X85137, NM004523 and U37426] , and a fragment of the KSP gene (TRIP5) has been described [Lee, et al., Mol Endocrinol., 9:243-54 (1995); GenBank accession number L40372]. Xenopus KSP homologs (Eg5), as well as Drosophila K-LP61 F/KRP 130 have been reported. Certain quinazolinones have recently been described as being inhibitors of KSP (PCT Publ. WO 01/30768, May 3, 2001).
Mitotic kinesins are attractive targets for the discovery and development of novel mitotic chemotherapeutics. Accordingly, it is an object of the present invention to provide compounds, methods and compositions useful in the inhibition of KSP, a mitotic kinesin.
SUMMARY OF THE INVENTION
The present invention relates to 4,5-dihydropyrazole derivatives, that are useful for treating cellular proliferative diseases, for treating disorders associated with KSP kinesin activity, and for inhibiting KSP kinesin. The compounds of the invention may be illustrated by the Formula I:
R3 R4 R5
N-N Rc
R1
DETAILED DESCRIPTION OF THE INVENTION The compounds of this invention are useful in the inhibition of mitotic kinesins and are illustrated by a compound of Formula I:
Figure imgf000004_0001
I
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
a is O or l; b is O or l; m is 0, 1, or 2; n is 0 to 2; u is 1, 2, 3, 4 or 5;
Rl is selected from:
1) (C=X)Cι-Cιo alkyl,
2) (C=X)aryl,
3) (C=X)C2-Cιo alkenyl, 4) (C=X)C2-Cιo alkynyl,
5) (C=X)C3-C8 cycloalkyl,
6) (C=X)heterocyclyl,
7) (C=X)NR7R8, 8) (C=X)OCι-Ci0 alkyl,
9) SO2NR7R8,
10) SO2Ci-Cιo alkyl,
11) SO2Cι-Cιo aryl, 12) SO2C1 -C10 heterocyclyl,
13) C1-C10 alkyl,
14) aryl,
15) heteroaryl,
16) (CH )u(C=O)Ci-Cio alkyl, 17) (CH2)u(C=O) NR7R8,
18) 3-pyrrolidinonyl, 3-piperidinonyl, 2-cyclopentanonyl, 2- cyclohexanonyl,
19) (C=O)(C=O)Ci-Cιo alkyl,
20) (C=O)(C=O)NR7R8, 21) (C=O)(C=O)O CI-CIO alkyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from: 1) C1-C10 alkyl,
2) aryl,
3) C2-C 10 alkenyl,
4) C2-C 10 alkynyl,
5) C1-C6 perfluoroalkyl, 6) C1-C6 aralkyl,
7) C1-C6 heteroaralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R4, R5 and R6 are independently selected from:
1) H,
2) C1-C10 alkyl, 3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl,
6) C 1 -C6 perfl uoroal ky 1 ,
7) C1-C6 aralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R3 and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(O)m, -N(R9)C(O)-, and -N(COR10)-;
is:
1) (C=O)aObCι-Cio alkyl,
2) (C=O)aObaryl,
3) C2-C 10 alkenyl,
4) C2-C 10 alkynyl,
5) (C=O)aOb heterocyclyl,
6) CO2H,
7) halo,
8) CN,
9) OH,
10) ObCι-C6 perfluoroalkyl,
Figure imgf000006_0001
12) S(O)mRa,
Figure imgf000006_0002
12) oxo,
13) CHO,
14) (N=O)R9R10, or
15) (C=O)aObC3-C8 cycloal said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R$; R8 is selected from:
1) (C=O)rOs(Cι-Cιo)alkyl, wherein r and s are independently 0 or 1,
2) Or(Cι-C3)perfluoroalkyl, wherein r is 0 or 1,
3) (Co-C6)alkylene-S(O)mRa, wherein m is 0, 1, or 2, 4) oxo,
5) OH,
6) halo,
7) CN,
8) (C=O)rOs(C2-Ciθ)alkenyl, 9) (C=O)rOs(C2-Cio)alkynyl,
10) (C=O)rOs(C3-C6)cycloalkyl,
11) (C=O)rOs(Co-C6)alkylene-aryl,
12) (C=O)rOs(Co-C6)alkylene-heterocyclyl,
13) (C=O)rOs(Co-C6)alkylene-N(Rb)2, 14) C(O)Ra,
15) (Co-C6)alkylene-CO2Ra,
16) C(O)H,
17) (Co-C6)alkylene-CO2H,
18) C(O)N(Rb)2, 19) S(O)mRa,
Figure imgf000007_0001
21) C(NH)NH2; said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from:
1) H,
2) (C=O)ObCι-Cιo alkyl,
3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl,
6) C1-C10 alkyl,
7) aryl, 8) C2-C 10 alkenyl,
9) C2-C ιo alkynyl,
10) heterocyclyl,
11) C3-C8 cycloalkyl, 12) SO2Ra, and
13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R8, or
R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
Ra is (Cι-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Cι-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCι-C6 alkyl, (C=O)Cι-C6 alkyl or S(O)2Ra;
X is selected from O and S;
Y, W and Z are independently selected from: a bond, C=O, C=S, S(O)n, CH(OH) and O.
In an embodiment of this invention, the compounds useful in the inhibition of mitotic kinesins are illustrated by the Formula I:
Figure imgf000008_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein a is O or l; b is O or l; m is 0, 1, or 2; n is 0 to 2; u is 2, 3, 4 or 5;
Rl is selected from:
1) (C=X)Ci-Cιo alkyl,
2) (C=X)aryl, 3) (C=X)C2-Cιo alkenyl,
4) (C=X)C2-Cio alkynyl,
5) (C=X)C3-C8 cycloalkyl,
6) (C=X)heterocyclyl,
7) (C=X)NR7R8, 8) (C=X)OCι-Cιo alkyl,
9) SO2NR7R8,
10) SO2Cι-Cιo alkyl,
11) SO2Cι-Cιo aryl,
12) SO2Cι-Cιo heterocyclyl, 13) Ci-Cio alkyl,
14) aryl,
15) heteroaryl,
16)
Figure imgf000009_0001
alkyl,
17) (CH2)u(C=O) NR7R8, 18) 3-pyrrolidinonyl, 3-piperidinonyl, 2-cyclopentanonyl, 2- cyclohexanonyl,
19) (C=O)(C=O)Ci-Cιo alkyl,
20) (C=O)(C=O)NR7R8,
21) (C=O)(C=O)O Ci-Cιo alkyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from:
1) Ci-Cio alkyl, 2) aryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl,
5) C1-C6 perfluoroalkyl, 6) C1-C6 aralkyl,
7) C1-C6 heteroaralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R , R5 and R6 are independently selected from:
1) H,
2) C1-C10 alkyl, 3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl,
6) C1-C6 perfluoroalkyl,
7) C1-C6 aralkyl, 8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(O)m, -N(R9)C(O)-, and -N(COR10)-;
R7 is: 1) (C=O)aObCι-Cio alkyl,
3) (C=O)aObaryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl,
5) (C=O)aOb heterocyclyl, 6) CO2H,
7) halo,
8) CN,
9) OH,
10) ODCι-C6 perfluoroalkyl,
Figure imgf000011_0001
12) S(O)mR ,
Figure imgf000011_0002
14) oxo,
15) CHO,
16) (N=O)R9R10, or
17) (C=O)aObC3-C8 cycloall said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R8;
R8 is selected from:
1)
Figure imgf000011_0003
wherein r and s are independently 0 or 1,
2) Or(Ci-C3)perfluoroalkyl, wherein r is 0 or 1,
3) (Co-C6)alkylene-S(O)mRa, wherein m is 0, 1, or 2,
4) oxo,
5) OH,
6) halo,
7) CN,
8) (C=O)rOs(C2-Cιo)alkenyl,
9) (C=O)rOs(C2-Cιo)alkynyl,
10) (C=O)ιOs(C3-C6)cycloalkyl,
11) (C=O)rOs(Co-C6)alkylene-aryl,
12) (C=O)rOs(Co-C6)alkylene-heterocyclyl,
13) (C=O)rOs(Co-C6)alkylene-N(Rb)2,
14) C(O)Ra,
15) (Co-C6)alkylene-CO2Ra,
16) C(O)H,
17) (Co-C6)alkylene-CO2H,
18) C(O)N(Rb)2,
Figure imgf000012_0001
said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from:
1) H,
2) (C=O)ObCi-Cio alkyl, 3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl,
6) C1-C10 alkyl,
7) aryl, 8) C2-C10 alkenyl,
9) C2-C10 alkynyl,
10) heterocyclyl,
11) C3-C8 cycloalkyl,
12) SO2Ra, and 13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R8, or
R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
Ra is (C -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Ci-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCi-C6 alkyl, (C=O)Cι-C6 alkyl or S(O)2Ra; X is selected from O and S;
Y, W and Z are independently selected from: a bond, C=O, C=S, S(O)n, CH(OH) and O.
A further embodiment of the present invention is illustrated by a compound of Formula II, or a pharmaceutically acceptable salt or stereoisomer;
Figure imgf000013_0001
wherein:
a is O or l; b is O or l: m is 0, 1, or 2; n is 0 to 2;
Rl is selected from:
1) (C=O)Cι-Cιo alkyl,
2) (C=O)aryl,
3) (C=O)C2-Cio alkenyl,
4) (C=O)C2-Cιo alkynyl,
5) (C=O)C3-C8 cycloalkyl,
6) (C=O)heterocyclyl,
7) (C=O)OCι-Cιo alkyl,
8) (C=O)NR7R8I
9) SO2NR7R8,
10) SO2Cι-Cιo alkyl,
11) SO2Ci-Ciθ aryl,
12) SO Cι-Cιo heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from: 1) Cl-CiO alkyl,
2) aryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl,
5) C1-C6 perfluoroalkyl, 6) C1-C6 aralkyl,
7) C3-C8 cycloalkyl, and
8) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R4, R5 and R6 are independently selected from:
1) H,
2) C1-C10 alkyl,
3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl,
6) C1-C6 perfluoroalkyl,
7) C1-C6 aralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, ; aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R7 is:
1) (C=O)aObCι-Cιo alkyl,
2) (C=O)aObaryl,
3) C2-C 10 alkenyl,
4) C2-C10 alkynyl,
5) (C=O)aOb heterocyclyl, 6) CO2H,
7) halo,
8) CN,
9) OH,
10) ObCi-C6 perfluoroalkyl,
Figure imgf000015_0001
12) S(O)mRa,
Figure imgf000015_0002
14) oxo,
15) CHO,
16) (N=O)R9R10, or
17) (C=O)aObC3-C8 cycloall said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one, two or three substituents selected from R8;
is selected from:
1)
Figure imgf000015_0003
wherein r and s are independently 0 or 1,
2) Or(Ci-C3)peι luoroalkyl, wherein r is 0 or 1,
3) oxo,
4) OH,
5) halo,
6) CN,
7) (C2-Clθ)alkenyl,
8) (C2-Ciθ)alkynyl,
9) (C=O)rOs(C3-C6)cycloalkyl,
10) (C=O)rOs(Co-C6)alkylene-aryl,
11) (C=O)rOs(Co-C6)alkylene-heterocyclyl,
12) (C=O)rOs(Co-C6)alkylene-N(Rb)2,
13) C(O)R ,
14) (Co-C6)alkylene-CO2Ra,
15) C(O)H,
16) (Co-C6)alkylene-CO2H, and
17) C(O)N(Rb)2,
18) S(O)mRa, and
Figure imgf000016_0001
said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H, CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from:
1) H,
2) (C=O)ObCι-Cιo alkyl, 3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl,
6) C1-C10 alkyl,
7) aryl, 8) C2-C10 alkenyl,
9) C2-C10 alkynyl,
10) heterocyclyl,
11) C3-C8 cycloalkyl,
12) SO2Ra and 13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one, two or three substituents selected from R8, or
R9 and R O can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one, two or three substituents selected from R8;
Ra is (Cι-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Cι-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCι-C6 alkyl, (C=O)Cι-C6 alkyl or S(O)2R Another embodiment is the compound of the Formula II described immediately above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:
Rl is selected from:
1) (C=O)Cι-Cιo alkyl,
2) (C=O)aryl,
3) (C=O)C3-C8 cycloalkyl,
4) (C=O)heterocyclyl, 5) (C=O)OCι-Cιo alkyl,
6) SO2NR7R8, and
7) SO2Ci-Cio alkyl, said alkyl, aryl, cycloalkyl, and heterocyclyl is optionally substituted with one, two or three substituents selected from R7;
R2 is selected from:
1) Ci-Cio alkyl,
2) aryl, and
3) heteroaryl, said alkyl, aryl and heteroaryl is optionally substituted with one or more substituents selected from R7;
R3 and R4 are independently selected from:
1) H, and 2) Ci-Cio alkyl, said alkyl is optionally substituted with one or more substituents selected from R7; and
R5 and R6 are independently selected from: 1) H,
2) Ci-Cio alkyl,
3) aryl, and
4) heterocyclyl, said alkyl, aryl and heterocyclyl is optionally substituted with one or more substituents selected from R7; and
R7, R8; R9; R10; Ra and Rb are as described immediately above.
In another embodiment of the compounds of Formula II hereinabove, one of R5 and R6 are selected from phenyl or pyridyl, optionally substituted with one or two substituents selected from R7.
Another embodiment is the compound of the Formula II described immediately above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R is phenyl, optionally substituted with one or two substituents selected from R7.
Specific examples of the compounds of the instant invention include:
l-acetyl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3-(2-chlorophenyl)-l-isobutyryl-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-acetyl-3-(2-chlorophenyl)-5-methyl-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-acetyl-3-(2,5-difluorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3- l-Acetyl-3-(2-fluorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3- l-Acetyl-3-(3-bromophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Acetyl-3-(2,3-dichlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3- l-Acetyl-3-(2,5-dichlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3- l-Propionyl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol 3-[l-Isobutyryl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Acetyl-3-(2-chlorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(3-chlorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(4-fluoro-3-hydroxyphenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl Jpiperazine
3-(2,5-difluorophenyl)- N,N-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-(3-hydroxyphenyl)-N,N-dimethyl-4,5-dihydro-lH-pyrazole- 1 -carboxamide
4-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl}morpholine
3-[3-(2,5-difluorophenyl)-l-(mo holin-4-ylcarbonyl)-4,5-dihydro-lH-pyrazol-5- yl]phenol
3-(2,5-difluorophenyl)- N,N-diethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole-l-carboxamide
3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH-pyrazole
3-[3-(2-fluoro-5-methylphenyl)-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH-pyrazol-
5-yl]phenol l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
3-[l-(azetidin-l-ylcarbonyl)-3-(5-chloro-2-fluorophenyl)-4,5-dihydro-lH-pyrazol-5- yl]phenol
l-(l-{ [3-(5-chloro-2-fluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl}piperidin-2-yl)-N,N-dimethylmethanamine
3-(2,5-difluorophenyl)-l,5-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole
l-acetyl-3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-N,5-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-N,N,5-trimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-ethyl-N-methyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-(hydroxymethyl)-N-methyl-5-phenyl-4,5-dihydro-lH- pyrazole- 1 -carboxamide
3-(2,5-difluorophenyl)-5-methyl-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro- lH-pyrazole
3-(2,5-difluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-methyl-5-phenyl- 4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-l,5-dimethyl-5-(3-hydroxyphenyl)-4,5-dihydro-lH-pyrazole
ethyl [3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazol-l-yl]acetate ethyl [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l-yl]acetate
ethyl 2-[3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazol-l- yljpropanoate
3-[3-(2,5-difluorophenyl)-l-(morpholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l-amine
3-(2,5-difluorophenyl)-l-(methylsulfonyl)-5-phenyl-4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-5-[3-(dimethylamino)propyl]-N-ethyl-5-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide
3-(2,5-difluorophenyl)-N-ethyl-5-{3-[(lH-imidazol-2-ylcarbonyl)amino]propyl}-5- phenyl-4,5-dihydro-lH-pyrazole-l-carboxamide
5-(2-aminoethyl)-3-(2,5-difluorophenyl)-N-methyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
5-(3-aminopropyl)-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
5-(3-aminobutyl)-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH-pyrazole- 1 -carboxamide
5-[3-(benzoylamino)propyl]-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide
3-(2,5-difluorophenyl)-5-[4-(dimethylamino)butyl]-N-ethyl-5-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide
3-(2,5-difluorophenyl)-5-[4-(dimethylnitroryl)but-l-yl]-N-ethyl-5-phenyl-4,5- dihydro-1 H-pyrazole- 1 -carboxamide 5-[4-(benzylamino)butyl]-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-5-{4-[(pyridin-4-ylmethyl)amino]butyl}- 4,5-dihydro-lH-pyrazole-l-carboxamide
3-[l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l-ol
3-[l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l-amine
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l-ol
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l-amine
3-[3-(5-chloro-2-fluorophenyl)-l-(moφholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro- lH-pyrazol-5-yl]propan-l-amine
N-{3-[3-(2,5-difluorophenyl)-l-(moφholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propyl } guanidine
5-(3-amino-3-phenylpropyl)-3-(2,5-difluorophenyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-lH-pyrazole-1-carboxamide
3-[3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH- pyrazol-5-yl]-l-methylpropylamine
3-[3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH- ρyrazol-5-yl]-l-(trifluoromethyl)propylamine
l-acetyl-3-(2,5-difluorophenyl)-4-methyl-5-phenyl-4,5-dihydro-lH-pyrazole or a pharmaceutically acceptable salt or stereoisomer thereof.
An embodiment of the compounds of the instant invention are:
l-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl}piperazine TFA salt,
3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-5-{4-[(pyridin-4-ylmethyl)amino]butyl}- 4,5-dihydro-lH-pyrazole-l-carboxamide bis TFA salt,
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l-amine hydrochloride salt, and
N-{3-[3-(2,5-difluorophenyl)-l-(moφholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propyl}guanidine TFA salt.
Particular examples of the compounds of the instant invention are:
3-[l-acetyl-3-(2,5-difluorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
Figure imgf000023_0001
3-(2,5-difluorophenyl)-5-(3-hydroxy-3-phenylpropyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-1 H-pyrazole- 1 -carboxamide
Figure imgf000024_0001
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro- 1 H-pyrazol-5-yl]propan- 1 -amine
Figure imgf000024_0002
or a pharmaceutically acceptable salt or stereoisomer thereof.
The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. In addition, the compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the invention, even though only one tautomeric structure is depicted.
When any variable (e.g. R7, R8, R95 et .) occurs more than one time in any constituent, its definition on each occurrence is independent at every other occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. Lines drawn into the ring systems from substituents represent that the indicated bond may be attached to any of the substitutable ring atoms. If the ring system is polycyclic, it is intended that the bond be attached to any of the suitable carbon atoms on the proximal ring only.
It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results. The phrase "optionally substituted with one or more substituents" should be taken to be equivalent to the phrase "optionally substituted with at least one substituent" and in such cases the preferred embodiment will have from zero to three substituents.
As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, Ci-Cio, as in "Ci-Cio alkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, "Ci-Cio alkyl" specifically includes methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, /-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on. The term "cycloalkyl" means a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2- ethyl-cyclopentyl, cyclohexyl, and so on.
When used in the phrases "C1-C6 aralkyl" and "C1-C6 heteroaralkyl" the term "C1-C6" refers to the alkyl portion of the moiety and does not describe the number of atoms in the aryl and heteroaryl portion of the moiety.
"Alkoxy" represents either a cyclic or non-cyclic alkyl group of indicated number of carbon atoms attached through an oxygen bridge. "Alkoxy" therefore encompasses the definitions of alkyl and cycloalkyl above. If no number of carbon atoms is specified, the term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic carbon-carbon double bonds may be present. Thus, "C2-C6 alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl groups include ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 10 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl radical having from 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl, butynyl, 3-methylbutynyl and so on. The straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
In certain instances, substituents may be defined with a range of carbons that includes zero, such as (Co-C6)alkylene-aryl. If aryl is taken to be phenyl, this definition would include phenyl itself as well as -CH2Ph, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph, and so on.
As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
The term heteroaryl, as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. The term "heterocycle" or "heterocyclyl" as used herein is intended to mean a 3- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. "Heterocyclyl" therefore includes the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof. Further examples of "heterocyclyl" include, but are not limited to the following: azetindinyl, benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4- dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, moφholinyl, thiomoφholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom.
In an embodiment, the term "heterocycle" or "heterocyclyl" as used herein is intended to mean a 5- to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. "Heterocyclyl" therefore includes, in this embodiment, the above mentioned heteroaryls, as well as dihydro and tetrathydro analogs thereof. Further examples of "heterocyclyl" include, but are not limited to the following: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, moφholinyl, thiomoφholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof.
Preferably, heterocycle is selected from 2-azepinone, benzimidazolyl, 2-diazapinone, imidazolyl, 2-imidazolidinone, indolyl, isoquinolinyl, moφholinyl, piperidyl, piperazinyl, pyridyl, pyrrolidinyl, 2-piperidinone, 2-pyrimidinone, 2- pyrollidinone, quinolinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, and thienyl.
As appreciated by those of skill in the art, "halo" or "halogen" as used herein is intended to include chloro, fluoro, bromo and iodo.
The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. For example, a (Cι-C6)alkyl may be substituted with one, two or three substituents selected from OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as moφholinyl, piperidinyl, and so on. In this case, if one substituent is oxo and the other is OH, the following are included in the definition: -C=O)CH2CH(OH)CH3, -(C=O)OH, -CH2(OH)CH2CH(O), and so on.
The moiety formed when, in the definition of R and R4 and R5 and R6 on the same carbon atom are combined to form -(CH2)u- is illustrated by the following:
Figure imgf000028_0001
In addition, such cyclic moieties may optionally include a heteroatom(s). Examples of such heteroatom-containing cyclic moieties include, but are not limited to:
Figure imgf000029_0001
In certain instances, R9 and RlO are defined such that they can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said heterocycle optionally substituted with one or more substituents selected from R8. Examples of the heterocycles that can thus be formed include, but are not limited to the following, keeping in mind that the heterocycle is optionally substituted with one or more (and preferably one, two or three) substituents chosen from R ;
Figure imgf000030_0001
Figure imgf000030_0002
Preferably Rl is selected from: (C=O)Cι-C6alkyl, (C=O)aryl, SO2C1- C6 alkyl, (C=O)OCι-C6alkyl, (C=O)NR7R8 and Sθ2aryl, optionally substituted with one to three substituents selected from R7. More preferably, Rl is acetyl, thioacetyl, sulfonamindo, N,N-dialkylamido or methylsulfonyl.
Preferably R is selected from aryl, optionally substituted with one to three substituents selected from R7. More preferably, R2 is phenyl, optionally substituted with one to three substituents selected from halo.
Also prefered is the definition of R3 and R as H.
Preferably R5 and R6 are selected from H, C1-C6 alkyl, aryl and heteroaryl, optionally substituted with one to two substituents selected from R7. More preferably, one of R5 and R6 is phenyl, optionally substituted with one substituents selected from R7, and the other of R5 and R6 is H or C1-C6 alkyl. Still more preferably, one of R5 and R6 is phenyl, optionally 3-substituted with one substituents selected from OH and NH2, and the other of R5 and R6 is H or C1-C6 alkyl.
Included in the instant invention is the free form of compounds of Formula I, as well as the pharmaceutically acceptable salts and stereoisomers thereof. Some of the specific compounds exemplified herein are the protonated salts of amine compounds. The term "free form" refers to the amine compounds in non-salt form. The encompassed pharmaceutically acceptable salts not only include the salts exemplified for the specific compounds described herein, but also all the typical pharmaceutically acceptable salts of the free form of compounds of Formula I. The free form of the specific salt compounds described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free forms may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the acid and base salts are otherwise pharmaceutically equivalent to their respective free forms for puφoses of the invention.
The pharmaceutically acceptable salts of the instant compounds can be synthesized from the compounds of this invention which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts of the basic compounds are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents. Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic or organic base. Thus, pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed by reacting a basic instant compound with an inorganic or organic acid. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy- benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like. When the compound of the present invention is acidic, suitable "pharmaceutically acceptable salts" refers to salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine, choline, N-N'-dibenzylethylenediamine, diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmoφholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, moφholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine, tromethamine and the like.
The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et al, "Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19.
It will also be noted that the compounds of the present invention are potentially internal salts or zwitterions, since under physiological conditions a deprotonated acidic moiety in the compound, such as a carboxyl group, may be anionic, and this electronic charge might then be balanced off internally against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen atom. The compounds of this invention may be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature or exemplified in the experimental procedures. The illustrative schemes below, therefore, are not limited by the compounds listed or by any particular substituents employed for illustrative puφoses. Substituent numbering as shown in the schemes does not necessarily correlate to that used in the claims and often, for clarity, a single substituent is shown attached to the compound where multiple substituents are allowed under the definitions of Formula I hereinabove. SCHEMES
As shown in Scheme A, condensation of a suitably substituted acetophenone A-l with a suitably substituted benzaldehyde A-2 provides the β- hydroxylcarbonyl intermediate A-3. Dehydration of A-3 with trifluoroacetic anhydride provides the α,β-unsaturated carbonyl compound A-4. Intermediate A-4 can then undergo reaction with hydrazine in the presence of a carboxylic acid A-5 to provide the N-acyldihydropyrazole A-6.
As shown in Scheme B, reaction of the intermediate A-4 with hydrazine may be performed without the carboyxlic acid and the intermediate can then be reacted with a variety of acetylating and electrophilic reagents. Scheme B also illustrates the preparation of the instant compounds by reaction of the A-4 intermediate with an N-substituted hydrazine to provide the instant compound B-3.
Scheme C illustrates preparation of the 5,5-disubstituted compound of the instant invention. As shown, reaction of the intermediate A-4 with, for example, a cuprate reagent provides intermediate C-l, which can then be dehydrated to provide the substituted chalcone C-2. Subsequent reaction with a hydrazine, as shown , provides the compound of the instant invention.
As shown in Scheme D, the reaction sequence shown in scheme A is generally applicable to preparing other compounds of the instant invention that have
2 5 6
R and R /R other than phenyl.
Scheme E illustrates preparation of the 5,5-disubstituted compound having a functional moiety (a hydroxy) which can then be further functionalized.
Scheme F shows a synthetic method for preparing compounds of the instant invention having 1,3,4,5-tetrasubstitution. The synthetic route is analogous to the method described in Scheme A but starting with a substituted acetophenone F-l.
SCHEME A
Figure imgf000034_0001
A-1 A-2
Figure imgf000034_0002
Figure imgf000034_0003
A-6 SCHEME B
Figure imgf000035_0001
Figure imgf000035_0002
Figure imgf000035_0003
SCHEME C
Figure imgf000036_0001
Figure imgf000036_0002
Figure imgf000036_0003
Figure imgf000036_0004
SCHEME D
O O
R + LiHMDS
OH, H^R£ THF
D-1 D-2
Figure imgf000037_0001
D-3
D-4
Figure imgf000037_0002
heat
SCHEME E
Figure imgf000038_0001
Figure imgf000038_0002
SCHEME F
Figure imgf000039_0001
F-1 A-2
Figure imgf000039_0002
F-4
Utilities
The compounds of the invention find use in a variety of applications. reciated by those in the art, mitosis may be altered in a variety of ways; that is, one can affect mitosis either by increasing or decreasing the activity of a component in the mitotic pathway. Stated differently, mitosis may be affected (e.g., disrupted) by disturbing equilibrium, either by inhibiting or activating certain components. Similar approaches may be used to alter meiosis. In a preferred embodiment, the compounds of the invention are used to modulate mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis. By "modulate" herein is meant altering mitotic spindle formation, including increasing and decreasing spindle formation. By "mitotic spindle formation" herein is meant organization of microtubules into bipolar structures by mitotic kinesins. By "mitotic spindle dysfunction" herein is meant mitotic arrest and monopolar spindle formation.
The compounds of the invention are useful to bind to and/or modulate the activity of a mitotic kinesin. In a preferred embodiment, the mitotic kinesin is a member of the bimC subfamily of mitotic kinesins (as described in U.S. Patent No. 6,284,480, column 5). In a further preferred embodiment, the mitotic kinesin is human KSP, although the activity of mitotic kinesins from other organisms may also be modulated by the compounds of the present invention. In this context, modulate means either increasing or decreasing spindle pole separation, causing malformation, i.e., splaying, of mitotic spindle poles, or otherwise causing moφhological perturbation of the mitotic spindle. Also included within the definition of KSP for these puφoses are variants and/or fragments of KSP. See PCT Publ. WO 01/31335: "Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell Proliferation States", filed Oct. 27, 1999, hereby incoφorated by reference in its entirety. In addition, other mitotic kinesins may be inhibited by the compounds of the present invention.
The compounds of the invention are used to treat cellular proliferation diseases. Disease states which can be treated by the methods and compositions provided herein include, but are not limited to, cancer (further discussed below), autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. It is appreciated that in some cases the cells may not be in a hyper- or hypoproliferation state (abnormal state) and still require treatment. For example, during wound healing, the cells may be proliferating "normally", but proliferation enhancement may be desired. Similarly, as discussed above, in the agriculture arena, cells may be in a "normal" state, but proliferation modulation may be desired to enhance a crop by directly enhancing growth of a crop, or by inhibiting the growth of a plant or organism which adversely affects the crop. Thus, in one embodiment, the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders or states.
The compounds, compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compounds, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaφosi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaφosi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions. The compounds of the instant invention may also be useful as antifungal agents, by modulating the activity of the fungal members of the bimC kinesin subgroup, as is described in U.S. Patent No. 6,284,480.
The compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
Additionally, the compounds of the instant invention may be administered to a mammal in need thereof using a gel extrusion mechanism (GEM) device, such as that described in USSN 60/144,643, filed on July 20, 1999, which is hereby incoφorated by reference.
As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absoφtion in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropyl-methylcellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate 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, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene- oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n- propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame. Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
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, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant. The pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
The sterile injectable preparation may also be a sterile injectable oil-in- water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
The injectable solutions or microemulsions may be introduced into a patient's blood stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this puφose 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 injectables. Compounds of Formula I may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed. (For puφoses of this application, topical application shall include mouth washes and gargles.)
The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
When a compound according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms.
In one exemplary application, a suitable amount of compound is administered to a mammal undergoing treatment for cancer. Administration occurs in an amount between about 0.1 mg kg of body weight to about 60 mg kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per day.
The instant compounds are also useful in combination with known therapeutic agents and anti-cancer agents. For example, the instant compounds are useful in combination with known anti-cancer agents. Combinations of the presently disclosed compounds with other anti-cancer or chemotherapeutic agents are within the scope of the invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita and S. Hellman (editors), 6th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Such anti-cancer agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, and agents that interfere with cell cycle checkpoints. The instant compounds are particularly useful when co-administered with radiation therapy.
In an embodiment, the instant compounds are also useful in combination with known anti-cancer agents including the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic agents, antiproliferative agents, prenyl -protein transferase inhibitors, HMG- CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors, and other angiogenesis inhibitors. "Estrogen receptor modulators" refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2- dimethyl- 1 -oxopropoxy-4-methyl-2- [4- [2-( 1 -piperidinyl)ethoxy] phenyl] -2H- 1 - benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4'-dihydroxybenzophenone-2,4- dinitrophenyl-hydrazone, and SH646.
"Androgen receptor modulators" refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5α-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
"Retinoid receptor modulators" refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, -difluoromethylornithine, ILX23-7553, trans-N-(4'- hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.
"Cytotoxic/cytostatic agents" refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, antimetabolites; biological response modifiers; hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors and ubiquitin ligase inhibitors. Examples of cytotoxic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-l,6- diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, l-(l l-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'- moφholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, and 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl- daunorubicin (see WO 00/50032). An example of a hypoxia activatable compound is tirapazamine.
Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).
Examples of microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'- norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, obulin isethionate, auristatin, cemadotin, RPR109881, BMS184476, vinflunine, cryptophycin, 2,3,4,5,6- pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L- proline-t-butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS 188797. In an embodiment the epothilones are not included in the microtubule inhibitors/microtubule-stabilising agents. Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzylidene- chartreusin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, l-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-lH, 12H- benzo[de]pyrano[3' ,4' :b,7]-indolizino[l ,2b]quinoline-10, 13(9H, 15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'- dimethylamino-2'-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy- 5,6-dimethyl-6H-pyrido[4,3-b]carbazole-l-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydro0xy-3,5- dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-l,3-dioxol- 6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]- phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5- (3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H- pyrazolo[4,5,l-de]acridin-6-one, N-[l-[2(diethylamino)ethylamino]-7-methoxy-9- oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4- carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,l-c] quinolin-7-one, and dimesna.
Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in PCT Publications WO 01/30768, WO 01/98278 and WO 02/078639, and pending U.S. Ser. Nos. 60/338,779 (filed December 6, 2001), 60/338,344 (filed December 6, 2001), 60/338,383 (filed December 6, 2001), 60/338,380 (filed December 6, 2001), 60/338,379 (filed December 6, 2001) and 60/344,453 (filed November 7, 2001). In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK, inhibitors of aurora kinase and inhibitors of Rab6-KJFL. "Antiproliferative agents" includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'-fluoromethylene-2'- deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L- manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4- oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][l,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl- L-glutamic acid, aminopterin, 5-flurouracil, alanosine, l l-acetyl-8-
(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-l,ll-diazatetracyclo(7.4.1.0.0)- tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-l-B-D-arabino furanosyl cytosine, 3- aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab. Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar. "HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3- methylglutaryl-CoA reductase. Compounds which have inhibitory activity for HMG- CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Patent 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The terms "HMG-CoA reductase inhibitor" and "inhibitor of HMG-CoA reductase" have the same meaning when used herein.
Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see U.S. Patent Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAVACHOL®; see U.S. Patent Nos. 4,346,227, 4,537,859, 4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Patent Nos. 5,354,772, 4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896), atorvastatin (LIPITOR®; see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. 5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996) and US Patent Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention. An illustration of the lactone portion and its corresponding open-acid form is shown below as structures I and π.
Figure imgf000051_0001
Lactone Open -Acid I II
In HMG-CoA reductase inhibitors where an open-acid form can exist, salt and ester forms may be formed from the open-acid, and all such forms are included within the meaning of the term "HMG-CoA reductase inhibitor" as used herein. In an embodiment, the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and in a further embodiment, simvastatin. Herein, the term "pharmaceutically acceptable salts" with respect to the HMG-CoA reductase inhibitor shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base, particularly those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium, as well as those salts formed from amines such as ammonia, ethylenediamine, N- methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, 1-p- chlorobenzyl-2-pyrrolidine-l'-yl-methylbenz-imidazole, diethylamine, piperazine, and tris(hydroxymethyl) aminomethane. Further examples of salt forms of HMG- CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate. Ester derivatives of the described HMG-CoA reductase inhibitor compounds may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy. "Prenyl-protein transferase inhibitor" refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-13 (GGPTase-JJ, also called Rab GGPTase). Examples of prenyl-protein transferase inhibiting compounds include (+)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-5-yl)methyl]-4-(3- chlorophenyl)- 1 -methyl-2( lH)-quinolinone, (-)-6-[amino(4-chlorophenyl)( 1 -methyl - lΗ-imidazol-5-yl)methyl]-4-(3-chlorophenyl)-l-methyl-2(lH)-quinolinone, (+)-6- [amino(4-chlorophenyl)(l-methyl-lΗ-imidazol-5-yl) methyl]-4-(3-chlorophenyl)-l- methyl-2(lH)-quinolinone, 5(S)-n-butyl-l-(2,3-dimethylphenyl)-4-[l-(4- cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, (S)-l-(3-chlorophenyl) -4-[l-(4- cyanobenzyl)-5-imidazolylmethyl]-5-[2-(ethanesulfonyl) methyl)-2-piperazinone, 5(S)-n-Butyl-l-(2-methylphenyl)-4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-2- piperazinone, l-(3-chlorophenyl) -4-[l-(4-cyanobenzyl)-2-methyl-5- imidazolylmethyl]-2-piperazinone, l-(2,2-diphenylethyl)-3-[N-(l-(4-cyanobenzyl)- lΗ-imidazol-5-ylethyl)carbamoyl]piperidine, 4-{ 5-[4-hydroxymethyl-4-(4- chloropyridin-2-ylmethyl)-piperidine-l-ylmethyl]-2-methylimidazol-l-ylmethyl} benzonitrile, 4-{5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-l-ylmethyl]-2- methylimidazol-l-ylmethyl}benzonitrile, 4-{3-[4-(2-oxo-2H-pyridin-l-yl)benzyl]- 3H-imidazol-4-ylmethyl}benzonitrile, 4-{3-[4-(5-chloro-2-oxo-2H-[l,2']bipyridin- 5 '-ylmethyl]-3H-imidazol-4-ylmethyl} benzonitrile, 4-{3-[4-(2-oxo-2H-[l,2'] bipyridin-5'-ylmethyl]-3H-imidazol-4-ylmethyl}benzonitrile, 4-[3-(2-oxo-l-phenyl- l,2-dihydropyridin-4-ylmethyl)-3H-imidazol-4-ylmethyl}benzonitrile, 18,19- dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-lH-imidazo[4,3- c][l,l l,4]dioxaazacyclo-nonadecine-9-carbonitrile, (±)-19,20-dihydro-19-oxo-5H- 18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo[-/]imidazo[4,3-
&][l,6,9,12]oxatriaza-cyclooctadecine-9-carbonitrile, 19,20-dihydro-19-oxo-5H,17H-
18,21-ethano-6,10:12,16-dimetheno-22H-imidazo[3,4- h] [ 1 ,8, 11 , 14]oxatriazacycloeicosine-9-carbonitrile, and (±)-l 9,20-dihydro-3-methyl- 19-oxo-5H-18,21-ethano-12,14-etheno-6,10-metheno-22H-benzo [J]imidazo[4,3- fc][l,6,9,12]oxa-triazacyclooctadecine-9-carbonitrile.
Other examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No. 5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Patent No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Patent No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp.1394-1401 (1999).
"Angiogenesis inhibitors" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-α, interleukin- 12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti- inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy- genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec, Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)- fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105: 141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp.963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).
Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80: 10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed August 8, 2001) and 60/349,925 (filed January 18, 2002).
"Agents that interfere with cell cycle checkpoints" refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents. Such agents include inhibitors of ATR, ATM, the Chkl and Chk2 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7-hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032. "Inhibitors of cell proliferation and survival signalling pathway" refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140 and WO 02/083138), inhibitors of Raf kinase (for example BAY-43- 9006 ), inhibitors of MEK (for example CI-1040 and PD-098059), inhibitors of mTOR (for example Wyeth CCI-779), and inhibitors of PI3K (for example LY294002).
The combinations with NSAID's are directed to the use of NSAID's which are potent COX-2 inhibiting agents. For puφoses of this specification an NSAJD is potent if it possess an IC50 for the inhibition of COX-2 of lμM or less as measured by cell or microsomal assays.
The invention also encompasses combinations with NSAID's which are selective COX-2 inhibitors. For puφoses of this specification NSAID's which are selective inhibitors of COX-2 are defined as those which possess a specificity for inhibiting COX-2 over COX-1 of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1 evaluated by cell or microsomal assays. Such compounds include, but are not limited to those disclosed in U.S. Patent 5,474,995, issued December 12, 1995, U.S. Patent 5,861,419, issued January 19, 1999, U.S. Patent 6,001,843, issued December 14, 1999, U.S. Patent 6,020,343, issued February 1, 2000, U.S. Patent 5,409,944, issued April 25, 1995, U.S. Patent 5,436,265, issued July 25, 1995, U.S. Patent 5,536,752, issued July 16, 1996, U.S. Patent 5,550,142, issued August 27, 1996, U.S. Patent 5,604,260, issued February 18, 1997, U.S. 5,698,584, issued December 16, 1997, U.S. Patent 5,710,140, issued January 20,1998, WO 94/15932, published July 21, 1994, U.S. Patent 5,344,991, issued June 6, 1994, U.S. Patent 5,134,142, issued July 28, 1992, U.S. Patent 5,380,738, issued January 10, 1995, U.S. Patent 5,393,790, issued February 20, 1995, U.S. Patent 5,466,823, issued November 14, 1995, U.S. Patent 5,633,272, issued May 27, 1997, and U.S. Patent 5,932,598, issued August 3, 1999, all of which are hereby incoφorated by reference.
Inhibitors of COX-2 that are particularly useful in the instant method of treatment are:
3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and
Figure imgf000055_0001
5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine;
Figure imgf000056_0001
or a pharmaceutically acceptable salt thereof.
General and specific synthetic procedures for the preparation of the COX-2 inhibitor compounds described above are found in U.S. Patent No. 5,474,995, issued December 12, 1995, U.S. Patent No. 5,861,419, issued January 19, 1999, and U.S. Patent No. 6,001,843, issued December 14, 1999, all of which are herein incoφorated by reference.
Compounds that have been described as specific inhibitors of COX-2 and are therefore useful in the present invention include, but are not limited to, the following:
Figure imgf000056_0002
Figure imgf000057_0001
or a pharmaceutically acceptable salt thereof.
Compounds which are described as specific inhibitors of COX-2 and are therefore useful in the present invention, and methods of synthesis thereof, can be found in the following patents, pending applications and publications, which are herein incoφorated by reference: WO 94/15932, published July 21, 1994, U.S. Patent No. 5,344,991, issued June 6, 1994, U.S. Patent No. 5,134,142, issued July 28, 1992, U.S. Patent No. 5,380,738, issued January 10, 1995, U.S. Patent No. 5,393,790, issued February 20, 1995, U.S. Patent No. 5,466,823, issued November 14, 1995, U.S. Patent No. 5,633,272, issued May 27, 1997, and U.S. Patent No. 5,932,598, issued August 3, 1999.
Compounds which are specific inhibitors of COX-2 and are therefore useful in the present invention, and methods of synthesis thereof, can be found in the following patents, pending applications and publications, which are herein incoφorated by reference: U.S. Patent No. 5,474,995, issued December 12, 1995, U.S. Patent No. 5,861,419, issued January 19, 1999, U.S. Patent No. 6,001,843, issued December 14, 1999, U.S. Patent No. 6,020,343, issued February 1, 2000, U.S. Patent No. 5,409,944, issued April 25, 1995, U.S. Patent No. 5,436,265, issued July 25, 1995, U.S. Patent No. 5,536,752, issued July 16, 1996, U.S. Patent No. 5,550,142, issued August 27, 1996, U.S. Patent No. 5,604,260, issued February 18, 1997, U.S. Patent No. 5,698,584, issued December 16, 1997, and U.S. Patent No. 5,710,140, issued January 20,1998.
Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2- butenyl)oxiranyl]-l-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino-l-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-lH-l,2,3-triazole-4- carboxamide,CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis[imino-N-methyl-4,2- pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(l,3-naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).
As used above, "integrin blockers" refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the 0 vβ3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the αvβ3 integrin and the CCvβ5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the αvβ6, «vβ8> ociβl, α2βl, ccsβl, α6βl and 0C6β4 integrins. The term also refers to antagonists of any combination of αvβ3, αvβ5, αγβ6' avP8> alβl> a2βl> «5βl> «όβl and αόβ4 integrins.
Some specific examples of tyrosine kinase inhibitors include N- (trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5- yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3- chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-moφholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11, 12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-lH- diindolo[l,2,3-fg:3',2',l'-kl]pyrrolo[3,4-i][l,6]benzodiazocin-l-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3- d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7- dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-l-phthalazinamine, and EMD 121974. Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods. For example, combinations of the instantly claimed compounds with PPAR-γ (i.e., PPAR-garnma) agonists and PPAR-δ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol Chem. 1999;274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice. (Arch. Ophthamol 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR- γ/α agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR- H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, GI262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3- trifluoromethyl-l,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)ρropoxy)- 2-ethylchromane-2-carboxylic acid (disclosed in USSN 60/235,708 and 60/244,697). Another embodiment of the instant invention is the use of the presently disclosed compounds in combination with gene therapy for the treatment of cancer. For an overview of genetic strategies to treating cancer see Hall et al (Am J Hum Genet 61:785-789, 1997) and Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Patent No. 6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998;5(8): 1105-13), and interferon gamma (/ Immunol 2000;164:217-222).
The compounds of the instant invention may also be administered in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins. Such MDR inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).
A compound of the present invention may be employed in conjunction with anti-emetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis, a compound of the present invention may be used in conjunction with other anti- emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S.Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712, an antidopaminergic, such as the phenothiazines (for example prochloφerazine, fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. For the treatment or prevention of emesis that may result upon administration of the instant compounds, conjunctive therapy with an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is preferred.
Neurokinin-1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Patent Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos. EP 0 360 390, 0 394 989, 0428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0 599 538, 0 610 793, 0 634402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications, which are incoφorated herein by reference.
In an embodiment, the neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is selected from: 2-(R)-(l- (R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo- lH,4H-l,2,4-triazolo)methyl)moφholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719,147.
A compound of the instant invention may also be administered with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous eythropoiesis receptor activator (such as epoetin alfa).
A compound of the instant invention may also be administered with an agent useful in the treatment of neutropenia. Such a neutropenia treatment agent is, for example, a hematopoietic growth factor which regulates the production and function of neutrophils such as a human granulocyte colony stimulating factor, (G- CSF). Examples of a G-CSF include filgrastim.
A compound of the instant invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.
Thus, the scope of the instant invention encompasses the use of the instantly claimed compounds in combination with a second compound selected from: 1) an estrogen receptor modulator,
2) an androgen receptor modulator,
3) retinoid receptor modulator,
4) a cytotoxic agent,
5) an antiproliferative agent, 6) a prenyl-protein transferase inhibitor,
7) an HMG-CoA reductase inhibitor,
8) an HIV protease inhibitor,
9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor, 11) a PPAR-γ agonists,
12) a PPAR-δ agonists,
13) an inhibitor of inherent multidrug resistance,
14) an anti-emetic agent,
15) an agent useful in the treatment of anemia, 16) an agent useful in the treatment of neutropenia,
17) an immunologic-enhancing drug,
18) an inhibitor of cell proliferation and survival signaling, and
19) an agent that interfers with a cell cycle checkpoint.
In an embodiment, the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal- derived growth factor, an inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metal loprotease) inhibitor, an integrin blocker, interferon-α, interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl- carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, or an antibody to VEGF. In an embodiment, the estrogen receptor modulator is tamoxifen or raloxifene.
Also included in the scope of the claims is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with radiation therapy and/or in combination with a compound selected from:
1) an estrogen receptor modulator,
2) an androgen receptor modulator, 3) a retinoid receptor modulator,
4) a cytotoxic agent,
5) an antiproliferative agent,
6) a prenyl-protein transferase inhibitor,
7) an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor,
9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor,
11) PPAR-γ agonists,
12) PPAR-δ agonists, 13) an inhibitor of inherent multidrug resistance,
14) an anti-emetic agent,
15) an agent useful in the treatment of anemia,
16) an agent useful in the treatment of neutropenia,
17) an immunologic-enhancing drug, 18) an inhibitor of cell proliferation and survival signaling, and
19) an agent that interfers with a cell cycle checkpoint.
And yet another embodiment of the invention is a method of treating cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with paclitaxel or trastuzumab.
The invention further encompasses a method of treating or preventing cancer that comprises administering a therapeutically effective amount of a compound of Formula I in combination with a COX-2 inhibitor. The instant invention also includes a pharmaceutical composition useful for treating or preventing cancer that comprises a therapeutically effective amount of a compound of Formula I and a compound selected from:
1) an estrogen receptor modulator,
2) an androgen receptor modulator, 3) a retinoid receptor modulator,
4) a cytotoxic agent,
5) an antiproliferative agent,
6) a prenyl-protein transferase inhibitor,
7) an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor,
9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor, and
11) a PPAR-γ agonist,
12) a PPAR-δ agonists; 13) an inhibitor of cell proliferation and survival signaling, and
14) an agent that interfers with a cell cycle checkpoint.
The invention further comprises the use of the instant compounds in a method to screen for other compounds that bind to KSP. To employ the compounds of the invention in a method of screening for compounds that bind to KSP kinesin, the KSP is bound to a support, and a compound of the invention (which is a mitotic agent) is added to the assay. Alternatively, the compound of the invention is bound to the support and KSP is added. Classes of compounds among which novel binding agents may be sought include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for candidate agents that have a low toxicity for human cells. A wide variety of assays may be used for this puφose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
The determination of the binding of the mitotic agent to KSP may be done in a number of ways. In a preferred embodiment, the mitotic agent (the compound of the invention) is labeled, for example, with a fluorescent or radioactive moiety and binding determined directly. For example, this may be done by attaching all or a portion of KSP to a solid support, adding a labeled mitotic agent (for example a compound of the invention in which at least one atom has been replaced by a detectable isotope), washing off excess reagent, and determining whether the amount of the label is that present on the solid support. Various blocking and washing steps may be utilized as is known in the art. By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal. In some embodiments, only one of the components is labeled. For
1 S example, the kinesin proteins may be labeled at tyrosine positions using I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using 125I for the proteins, for example, and a fluorophor for the mitotic agents.
The compounds of the invention may also be used as competitors to screen for additional drug candidates. "Candidate bioactive agent" or "drug candidate" or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactivity. They may be capable of directly or indirectly altering the cellular proliferation phenotype or the expression of a cellular proliferation sequence, including both nucleic acid sequences and protein sequences. In other cases, alteration of cellular proliferation protein binding and/or activity is screened. Screens of this sort may be performed either in the presence or absence of microtubules. In the case where protein binding or activity is screened, preferred embodiments exclude molecules already known to bind to that particular protein, for example, polymer structures such as microtubules, and energy sources such as ATP. Preferred embodiments of assays herein include candidate agents which do not bind the cellular proliferation protein in its endogenous native state termed herein as "exogenous" agents. In another preferred embodiment, exogenous agents further exclude antibodies to KSP.
Candidate agents can encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
Competitive screening assays may be done by combining KSP and a drug candidate in a first sample. A second sample comprises a mitotic agent, KSP and a drug candidate. This may be performed in either the presence or absence of microtubules. The binding of the drug candidate is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to KSP and potentially modulating its activity. That is, if the binding of the drug candidate is different in the second sample relative to the first sample, the drug candidate is capable of binding to KSP. In a preferred embodiment, the binding of the candidate agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to KSP, such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the candidate agent and the binding moiety, with the binding moiety displacing the candidate agent.
In one embodiment, the candidate agent is labeled. Either the candidate agent, or the competitor, or both, is added first to KSP for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between about 4 and about 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding. In a preferred embodiment, the competitor is added first, followed by the candidate agent. Displacement of the competitor is an indication the candidate agent is binding to KSP and thus is capable of binding to, and potentially modulating, the activity of KSP. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate agent is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the candidate agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate the candidate agent is bound to KSP with a higher affinity. Thus, if the candidate agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate the candidate agent is capable of binding to KSP.
It may be of value to identify the binding site of KSP. This can be done in a variety of ways. In one embodiment, once KSP has been identified as binding to the mitotic agent, KSP is fragmented or modified and the assays repeated to identify the necessary components for binding.
Modulation is tested by screening for candidate agents capable of modulating the activity of KSP comprising the steps of combining a candidate agent with KSP, as above, and determining an alteration in the biological activity of KSP. Thus, in this embodiment, the candidate agent should both bind to KSP (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods and in vivo screening of cells for alterations in cell cycle distribution, cell viability, or for the presence, moφohology, activity, distribution, or amount of mitotic spindles, as are generally outlined above.
Alternatively, differential screening may be used to identify drug candidates that bind to the native KSP, but cannot bind to modified KSP.
Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non- specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
These and other aspects of the invention will be apparent from the teachings contained herein.
ASSAYS
The compounds of the instant invention described in the Examples were tested by the assays described below and were found to have kinesin inhibitory activity. Other assays are known in the literature and could be readily performed by those of skill in the art (see, for example, PCT Publication WO 01/30768, May 3, 2001, pages 18-22).
I. Kinesin ATPase In Vitro Assay
Cloning and expression of human poly-histidine tagged KSP motor domain (KSP(367H))
Plasmids for the expression of the human KSP motor domain construct were cloned by PCR using a pBluescript full length human KSP construct (Blangy et al., Cell, vol.83, ppl 159-1169, 1995) as a template. The N-terminal primer 5'- GCAACGATTAATATGGCGTCGCAGCCAAATTCGTCTGCGAAG (SEQ.ID.NO.: 1) and the C-terminal primer 5'-GCAACGCTCGAGTCAGTGAT GATGGTGGTGATGCTGATTCACTTCAGGCTTATTCAATAT (SEQ.ID.NO. : 2)were used to amplify the motor domain and the neck linker region. The PCR products were digested with Asel and Xhol, ligated into the Ndel/Xhol digestion product of pRSETa (Invitrogen) and transformed into E. coli BL21 (DE3).
Cells were grown at 37°C to an OD6oo of 0.5. After cooling the culture to room temperature expression of KSP was induced with lOOμM IPTG and incubation was continued overnight. Cells were pelleted by centrifugation and washed once with ice-cold PBS. Pellets were flash-frozen and stored -80°C.
Protein Purification
Cell pellets were thawed on ice and resuspended in lysis buffer (50mM K-HEPES, pH 8.0, 250mM KC1, 0.1% Tween, lOmM imidazole, 0.5mM Mg-ATP, ImM PMSF, 2mM benzimidine, lx complete protease inhibitor cocktail (Roche)). Cell suspensions were incubated with lmg/ml lysozyme and 5mM β-mercaptoethanol on ice for 10 minutes, followed by sonication (3x 30sec). All subsequent procedures were performed at 4°C. Lysates were centrifuged at 40,000x g for 40 minutes. Supernatants were diluted and loaded onto an SP Sepharose column (Pharmacia, 5ml cartridge) in buffer A (50mM K-HEPES, pH 6.8, ImM MgCl2, ImM EGTA, lOμM Mg-ATP, ImM DTT) and eluted with a 0 to 750mM KC1 gradient in buffer A. Fractions containing KSP were pooled and incubated with Ni-NTA resin (Qiagen) for one hour. The resin was washed three times with buffer B (Lysis buffer minus PMSF and protease inhibitor cocktail), followed by three 15-minute incubations and washes with buffer B. Finally, the resin was incubated and washed for 15 minutes three times with buffer C (same as buffer B except for pH 6.0) and poured into a column. KSP was eluted with elution buffer (identical to buffer B except for 150mM KC1 and 250mM imidazole). KSP-containing fractions were pooled, made 10% in sucrose, and stored at -80°C.
Microtubules are prepared from tubulin isolated from bovine brain. Purified tubulin (> 97% MAP-free) at 1 mg/ml is polymerized at 37°C in the presence of 10 μM paclitaxel, 1 mM DTT, 1 mM GTP in BRB80 buffer (80 mM K-PIPES, 1 mM EGTA, 1 mM MgCl2 at pH 6.8). The resulting microtubules are separated from non-polymerized tubulin by ultracentrifugation and removal of the supernatant. The pellet, containing the microtubules, is gently resuspended in 10 μM paclitaxel, 1 mM DTT, 50 μg/ml ampicillin, and 5 μg ml chloramphenicol in BRB80.
The kinesin motor domain is incubated with microtubules, 1 mM ATP (1:1 MgCl2: Na-ATP), and compound at 23°C in buffer containing 80 mM K-HEPES (pH 7.0), 1 mM EGTA, 1 mM DTT, 1 mM MgCl2, and 50 mM KC1. The reaction is terminated by a 2-10 fold dilution with a final buffer composition of 80 mM HEPES and 50 mM EDTA. Free phosphate from the ATP hydrolysis reaction is measured via a quinaldine red/ammonium molybdate assay by adding 150 μl of quench C buffer containing a 2:1 ratio of quench A:quench B. Quench A contains 0.1 mg/ml quinaldine red and 0.14% polyvinyl alcohol; quench B contains 12.3 mM ammonium molybdate tetrahydrate in 1.15 M sulfuric acid. The reaction is incubated for 10 minutes at 23°C, and the absorbance of the phospho-molybdate complex is measured at 540 nm.
The compounds 1-7, 2-1, 3-3, 4-5, 5-6, 6-2, 7-7, 8-1, 9-2, 10-2, 11-2 and 12-2 and the compounds shown in Tables 1-6 in the Examples were tested in the above assay and found to have an IC50 ≤ 50μM.
II. Cell Proliferation Assay
Cells are plated in 96-well tissue culture dishes at densities that allow for logarithmic growth over the course of 24, 48, and 72 hours and allowed to adhere overnight. The following day, compounds are added in a 10-point, one-half log titration to all plates. Each titration series is performed in triplicate, and a constant DMSO concentration of 0.1% is maintained throughout the assay. Controls of 0.1% DMSO alone are also included. Each compound dilution series is made in media without serum. The final concentration of serum in the assay is 5% in a 200 μL volume of media. Twenty microliters of Alamar blue staining reagent is added to each sample and control well on the titration plate at 24, 48, or 72 hours following the addition of drug and returned to incubation at 37°C. Alamar blue fluorescence is analyzed 6-12 hours later on a CytoFluor II plate reader using 530-560 nanometer wavelength excitation, 590 nanometer emission.
A cytotoxic EC50 is derived by plotting compound concentration on the x-axis and average percent inhibition of cell growth for each titration point on the y-axis. Growth of cells in control wells that have been treated with vehicle alone is defined as 100% growth for the assay, and the growth of cells treated with compounds is compared to this value. Proprietary in-house software is used to calculate percent cytotoxicity values and inflection points using logistic 4-parameter curve fitting. Percent cytotoxicity is defined as:
% cytotoxicity:(FluorescenceControi) - (Flourescencesampιe) xlOOx (Fluorescencecontroi) '
The inflection point is reported as the cytotoxic EC50.
III. Evaluation of mitotic arrest and apoptosis by FACS FACS analysis is used to evaluate the ability of a compound to arrest cells in mitosis and to induce apoptosis by measuring DNA content in a treated population of cells. Cells are seeded at a density of 1.4x10° cells per 6cm2 tissue culture dish and allowed to adhere overnight. Cells are then treated with vehicle
(0.1% DMSO) or a titration series of compound for 8-16 hours. Following treatment, cells are harvested by trypsinization at the indicated times and pelleted by centrifugation. Cell pellets are rinsed in PBS and fixed in 70% ethanol and stored at
4°C overnight or longer.
For FACS analysis, at least 500,000 fixed cells are pelleted and the
70% ethanol is removed by aspiration. Cells are then incubated for 30 min at 4°C with RNase A (50 Kunitz units/ml) and propidium iodide (50 μg/ml), and analyzed using a
Becton Dickinson FACSCaliber. Data (from 10,000 cells) is analyzed using the
Modfit cell cycle analysis modeling software (Verity Inc.).
An EC50 for mitotic arrest is derived by plotting compound concentration on the x-axis and percentage of cells in the G2/M phase of the cell cycle for each titration point (as measured by propidium iodide fluorescence) on the y-axis. Data analysis is performed using the SigmaPlot program to calculate an inflection point using logistic 4-parameter curve fitting. The inflection point is reported as the EC50 for mitotic arrest. A similar method is used to determine the compound EC50 for apoptosis. Here, the percentage of apoptotic cells at each titration point (as determined by propidium iodide fluorescence) is plotted on the y-axis, and a similar analysis is carried out as described above.
VI. Immunofluorescence Microscopy to Detect Monopolar Spindles Methods for immunofluorescence staining of DNA, tubulin, and pericentrin are essentially as described in Kapoor et al. (2000) J. Cell Biol. 150: 975-988. For cell culture studies, cells are plated on tissue-culture treated glass chamber slides and allowed to adhere overnight. Cells are then incubated with the compound of interest for 4 to 16 hours. After incubation is complete, media and drug are aspirated and the chamber and gasket are removed from the glass slide. Cells are then permeabilized, fixed, washed, and blocked for nonspecific antibody binding according to the referenced protocol. Paraffin-embedded tumor sections are deparaffinized with xylene and rehydrated through an ethanol series prior to blocking. Slides are incubated in primary antibodies (mouse monoclonal anti-α-tubulin antibody, clone DM1 A from Sigma diluted 1:500; rabbit polyclonal anti-pericentrin antibody from Covance, diluted 1:2000) overnight at 4°C. After washing, slides are incubated with conjugated secondary antibodies (FITC-conjugated donkey anti- mouse IgG for tubulin; Texas red-conjugated donkey anti-rabbit IgG for pericentrin) diluted to 15μg/ml for one hour at room temperature. Slides are then washed and counterstained with Hoechst 33342 to visualize DNA. Immunostained samples are imaged with a lOOx oil immersion objective on a Nikon epifluorescence microscope using Metamoφh deconvolution and imaging software.
EXAMPLES
Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be illustrative of the invention and not limiting of the reasonable scope thereof. SCHEME 1
Figure imgf000072_0001
Step 1: 3-r3-(benzyloxy)phenyll-l-(2-chlorophenyl)prop-2-en-l-one (1-4)
To a solution of 2'-chloroacetophenone (1-1) (1.26mL, 9.70mmol) in 40 mL of THF at -78°C was slowly added 10.7 mL (10.7mmol) of a 1M LiHMDS solution in THF. After stirring for lh at -78°C, a solution of 2.05g (9.70mmol) of 3- benzyloxy-benzaldehyde (1-2) in 8 mL of THF was added, and stirring was continued at that temperature for an additional hour. The mixture was then dumped into a separatory funnel containing 100 mL of saturated aqueous NHjCl and extracted twice with 100 mL of EtOAc. The organic phases were combined, washed with 100 mL of brine, and dried over Na2SO4. After filtering off the drying agent, the solvent was removed on a rotary evaporator, and the residue was dissolved in 50 mL of CH2C1 . After cooling to -78°C, 4 mL of triethylamine and 2 mL of trifluoroacetic anhydride were added sequentially, and the mixture was allowed to warm to rt and stir for 12h. The reaction was then dumped into a separatory funnel with 100 mL of 1M HC1, the layers were separated, and the aqueous phase extracted again with CH C12. The organic layers were combined, washed again with 1 M HC1, washed with water, and dried over Na2SO4. After concentration, the crude material was purified by chromatography on silica gel with a gradient of 0 to 40% EtOAc in hexanes over 45 min to provide Λ as a viscous yellow oil. Data for L4: 1HNMR (500 MHz, CDC13) δ 7.5 - 7.0 (m, 15H) 5.1 (s, 2H) ppm.
Step 2: l-(2-chlorophenyl)-3-(hvdroxyphenyl)prop-2-en-l-one (1-5) To a solution of 740 mg (2.12mmol) of L in 15 mL of CH2C12 at
-78°C was added dropwise 2.75 mL (2.75mmol) of a 1M solution of BBr3 in CH2C12. After stirring for 30 min at that temperature, 1 mL of MeOH was added, and the mixture was dumped into water, extracted twice with 50 mL of CH2C1 , washed again with water, and dried over Na2SO . After concentration, the residue was purified by column chromatography on silica gel with a gradient of 2 to 70% EtOAc in hexanes over 30 min to provide L5 as a beige solid. Data for 1^5: 1HNMR (500 MHz, CDC13) δ 7.5 - 7.3 (m, 5H), 7.25 (m, 1H), 7.2 - 7.0 (m, 3H), 6.9 (m, 1H), 5.1 (bs, 1H) ppm.
Step 3: 3-[l-acetyl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol (1 Z)
To a solution of 120mg (0.46mmol) of chalcone 1^5 in 4 mL of acetic acid was added 50 μL (0.93mmol) of hydrazine hydrate. The reaction was then placed in an oil bath at 110°C for 24h. After cooling to rt, the solvents were removed on a rotary evaporator, the residue was dissolved in 50 mL of CH2C12, washed twice with aqueous NaHCO3, dried over Na2SO , and concentrated. The residue was then purified by column chromatography on silica gel with a gradient of 5 to 75% EtOAc in hexanes over 30 min to provide 7 as a fluffy white solid. Data for L7: 'HNMR (500 MHz, CDC13) δ 7.75 (m, 1H), 7.45 (m 1H), 7.4 - 7.3 (m, 2H), 7.2 (m, 1H), 6.8 (d, 1H), 6.7 (m, 2H), 5.5 (m, 1H), 3.9 (m, 1H), 3.3 (m, 1H), 2.4 (s, 3H) ppm. HRMS (ES) calc'd M + H for Cι7Hι5ClN2O2: 315.0895. Found: 315.0904.
SCHEME 2
Figure imgf000074_0001
1-5
3-[3-(2-chlorophenyl)-l-isobutyryl-4,5-dihydro-lH-pyrazol-5-yllphenol (2-1)
To a solution of 150mg (0.58mmol) of chalcone L5 in 4 mL of CH2C12 was added 30 μL (0.64mmol) of hydrazine hydrate. The mixture was then heated in a microwave reactor at 150°C for 5 min. After cooling to 0°C, 268 μL (2.9mmol) of isobutyric acid was added, followed by 135 mg (0.70mmol) of EDCI (l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride). The reaction was then warmed to rt and allowed to stir for 18h. The mixture was dumped into a separatory funnel, washed twice with 10% aqueous citric acid, twice with NaHCO3, dried over Na2SO , and concentrated. The residue was purified by column chromatography on silica gel with a gradient of 0 to 75% EtOAc in hexanes over 30 min to provide 2Λ as a pale orange film. Data for 2 : 'HNMR (500 MHz, CDC13) δ 7.7 (m, 1H), 7.45 (m, 1H), 7.3 (m, 2H), 7.1 (m, 1H), 6.7 (m, 2H), 6.65 (m, 1H), 6.55 (bs, 1H), 5.5 (m, 1H), 3.9 (m, 1H), 3.5 (m, 1H), 3.3 (m, 1H), 1.3 (m, 6H) ppm. HRMS (ES) calc'd M + H for Cι9H19ClN2O2: 343.1208. Found: 343.1213. SCHEME 3
Figure imgf000075_0001
Figure imgf000075_0002
3-ri-acetyl-3-(2-chlorophenyl)-5-methyl-4,5-dihydro-lH-pyrazol-5-yllphenol (3-3)
To a suspension of Cul (721 mg, 3.78mmol) in 15mL of THF at -78°C was added 2.52 mL (7.57mmol) of a 3M solution of MeMgBr in Et O. The mixture was stirred for 30 min, warmed slowly to -30°C, cooled back to -78°C, and a solution of 1.2 g (3.44mmol) of chalcone L4 in 10 mL of THF was added dropwise. The reaction was then warmed to 0°C and stirred for 30 min, at which time TLC analysis indicated complete reaction. After carefully quenching with saturated NHjCl, the mixture was extracted twice with EtOAc, washed with brine, and dried over Na2SO . Following concentration, the product was purified by chromatography on silica gel with a gradient of 0 to 45% EtOAc in hexanes over 45 min to provide 3A as a yellow oil, contaminated with a significant amount of another compound assumed to be the 1,2-addition product. This crude material was then dissolved in 15mL of THF, cooled to -78°C, and 2.26mL (2.26mmol) of a 1M solution of LiHMDS in THF was added dropwise. After stirring for lh, a solution of 535mg (2.26mmol) of PhSeBr in 2 mL of THF was added, and stirring was continued for 30 min. The reaction was then dumped into saturated NHjCl, extracted twice with EtOAc, washed with brine and dried. This crude material was dissolved in 15mL of Et2O, and after the addition of 2 mL of H2O and 500 μL of HO Ac, 2 mL of 30% aqueous H2O2 was added and the mixture was stirred for 20 min. The mixture was then dumped into a separatory funnel with saturated aqueous NaHCO3, extracted twice with Et2O, washed with brine, and dried over MgSO4. After concentration, the crude material was purified by chromatography on silica gel with a gradient of 0 to 35% EtOAc in hexanes over 45 min to provide 3^2 as a yellow oil, which was characterized only by LC-MS to have the desired m z = 362. To 50 mg (0.14mmol) of 3^2 dissolved in 5 mL of CH2C12 at - 78°C was added 210 μL (0.21mmol) of a 1M solution of BBr3 in CH2C12. After stirring for 20 min, 500 μL of MeOH was added, and the resultant mixture was dumped into water, extracted twice with CH2C12, washed twice with water, and dried over Na2SO4. After concentration, this material was dissolved in 2.5 mL of CH2C1 , 22 μL (0.45mmol) of hydrazine hydrate was added, and the mixture was heated at 150°C in a microwave reactor for 5 min. After cooling the reaction to -78°C, 210 mg (1.5mmol) of K2CO3 and 54 μL (0.75mmol) of acetyl chloride were added, and the reaction was allowed to warm to rt and stir for 12h. The mixture was then dumped into a separatory funnel with 10% aqueous citric acid, extracted twice with CH2C12, washed with NaHCO3, and dried over Na SO4. After concentration, the residue was purified by column chromatography on silica gel with a gradient of 2 to 75% EtOAc in hexanes over 45 min to provide 3^3 as an off-white taffy. Data for βθ: 1HNMR (500 MHz, CDC13) δ 7.7 (m, 1H), 7.4 (m, 1H), 7.3 (m, 2H), 7.1 (m, 1H), 6.8 (m, 1H), 6.7 (m, IH), 6.6 (m, IH), 5.9 (s, IH), 3.6 (m, 2H), 2.4 (s, 3H), 2.0 (s, 3H) ppm. HRMS (ES) calc'd M + H for Cι8Hι7ClN2O2: 329.1051. Found: 329.1045.
SCHEME 4
Figure imgf000077_0001
4-3
Figure imgf000077_0002
4-4
Figure imgf000077_0003
3-ri-acetyl-3-(2,5-difluorophenyl)-4,5-dihvdro-lH-pyrazol-5-vHphenol (4-5) Compound 4^5 was prepared starting from 2,5-difluoroacetophenone (4-1, available in bulk from Marshallton Research Laboratories) and 3- benzyloxybenzaldehyde (4-2) by a process analogous to that described in Scheme 1 for j_. Data for 4^5: 1HNMR (500 MHz, CDC13) δ 7.85 (m, IH), 7.2 - 7.0 (m, 3H), 6.8 (m, IH), 6.7 (m, 2H), 5.5 (m, IH), 5.3 (s, IH), 3.8 (m, IH), 3.3 (m, IH), 2.4 (s, 3H) ppm. HRMS (FT/ICR) calc'd M + H for Cι7Hι4F2N2O2: 317.1096. Found: 317.1108.
Chiral resolution of racemic 4-5 was achieved by HPLC chromatography (Chiralcel AD 5 x 50 cm column; eluting with 80% hexanes (+0.1% TFA)/20% ethanol at 75 mL/min) to give the faster eluting (+)-enantiomer Rτ = 20.7 min and slower eluting(-)-enantiomer RT = 34.5 min.
The compounds of the invention illustrated below in Table 1 were prepared by the synthetic methods described hereinabove, but substituting the appropriate acetophenones, phenyl aldehydes and carboxylic acids for the corresponding reagents utilized in the above examples:
TABLE 1
Figure imgf000079_0001
R sub R 7a R 7b
HRMS (calc'd) HRMS (found)
CH, 2-F 3-OH 299.1 191 299.1195
CH, 3-Br 3-OH 359.0390 359.0399
CH, 2,3-diCI 3-OH 349.0505 349.0506
CH, 2,5-diCI 3-OH 349.0505 349.0511
Et 2-CI 3-OH 329.1052 329.1055
i-Pr 2-CI 3-OH 343.1208 343.1213
CH3 2-CI H 299.0946 299.0949
CH3 3-CI H 299.0946 299.0954
CH3 2, 5-diF H 301.1 147 301.1154
CH3 2, 5-diF 4-F, 3-OH 335.1002 335.1006
CF3 2,5-diF 3-OH 371.0814 371.0810 tBu 2,5-diF H 343.1617 343.1615 SCHEME 5
Figure imgf000080_0001
Figure imgf000080_0002
4-nitrophenyl 3-(2.5-difluorophenyl)-5-phenyl-4,5-dihvdro-lH-pyrazole-l- carboxylate (5-3)
To a solution of 200mg (0.82 mmol) of chalcone 5A in CH C12 was added 50 μL (1.0 mmol) of hydrazine hydrate. The reaction was heated in a microwave reactor for 5 min at 150°C, and the solvents were then removed on a rotary evaporator. The residue was dissolved in THF, 170mg (1.23 mmol) of K2CO3 was added, followed by 496mg (2.46 mmol) of 4-nitrophenyl cnloroformate 5^2, and the reaction was stirred overnight. After quenching with 1M HC1, the mixture was dumped into a separatory funnel and extracted with 2 x EtOAc. The organic phases were combined, washed with brine, and dried over MgSO . After concentration, the residue was purified by column chromatography on silica gel with EtOAc/hexanes, then triturated with EtOAc/hexanes to provide 5^3 as a white solid. Data for 5-3: 1HNMR (500 MHz, CDC13) δ 8.1 (bs, 2H), 7.9 (m, IH), 7.5 - 7.0 (m, 9H), 5.6 (bs, IH), 4.0 (m, IH), 3.5 (m, 2H) ppm. HRMS (ES) calc'd M + H f or C22Hl5F2N3O4: 424.1104. Found: 424.1106.
l-| [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yllcarbonyllpiperazine (5-6)
To 80mg (0.19 mmol) of carbamate 5^3 in DMF was added 42mg (0.23 mmol) of amine 5 . The reaction was then placed in a microwave reactor at 200°C for 5 min. After cooling, the reaction was partitioned between EtOAc and saturated NaHCO3, the phases were separated, and the organic phase was washed with brine, dried over MgSO4, and concentrated by rotary evaporation. The residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide 5z5 as a white foam. Data for 5^5: HRMS (ES) calc'd M + Na for C25H28F2N4O3:
493.2021. Found: 493.2024. 5^ (75mg, 0.16 mmol) was dissolved in CH2C12 and 91 mg (0.8 mmol) of trifluoroacetic acid was added, and the mixture was stirred for 30 min. The solvents were removed by rotary evaporation, and the residue was purified by reverse phase HPLC and lyophilized to provide the TFA salt of 5^ as a colorless gum. Data for φ HRMS (ES) calc'd M + H for C20H20F2N4O: 371.1683. Found: 371.1674.
The compounds of the invention illustrated below in Table 2 were prepared by the synthetic methods described hereinabove, but substituting the appropriate acetophenones, phenyl aldehydes, carboxylic acids and amines for the corresponding reagents utilized in the above examples:
TABLE 2
Figure imgf000083_0001
R sub R 7a R 7b
HRMS (calc'd) HRMS (found)
NMe, F F H 330.1413 330.1413
NMe, F F OH 346.1362 346.1365
Figure imgf000083_0002
NEt5 F H 358.1726 358.1726
NH F H 302.1 100 302.1 102
F H 378.1388 378.1393
— N
Me OH 368.1769 368.1773
F H 342.1413 342.1413
— N
Cl OH 374.1066 374.1071
Figure imgf000083_0003
SCHEME 6
Figure imgf000084_0001
3-(2,5-difluorophenyl)- 5-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole (6-2)
To a solution of 130mg (0.50 mmol) of chalcone 6_T in EtOH was added 80 μL (1.5 mmol) of methylhydrazine and several drops of HO Ac. The reaction was heated in a microwave reactor for 20 min at 150°C, and the solvents were removed on a rotary evaporator. The residue was dissolved in CH2C12, washed with saturated NaHCO , washed with water, and dried over Na2SO . After concentration, the residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide 6X2 as a colorless oil. Data for - 'HNMR (500 MHz, CDC13) δ 7.6-7.5 (m, 3H), 7.35 (m, 2H), 7.3 (m, IH), 7.0-6.9 (m, 2H), 3.3 (m, 2H), 2.9 (s, 3H) ppm. HRMS (ES) calc'd M + H for Cι7H16F2N2: 287.1355. Found: 287.1362.
SCHEME 7
Figure imgf000085_0001
Figure imgf000085_0002
Figure imgf000086_0001
Figure imgf000086_0002
Step 1: 6-{ [tert-butyl(dimethyl)silyl]oxy}-l-(2,5-difluorophenyl)hex-2-yn-l- one (7-3)
To a solution of 4.0g (20.2 mmol) of 7^2 in THF at -78°C was added dropwise 8.5 mL (21.2 mmol) of a 2.5M solution of BuLi in hexanes. After stirring for lh, 4.0g (20.2 mmol) of Weinreb amide 7^1 (made from the acid chloride and N,O-dimethylhydroxylamine) in THF was added dropwise, the solution was allowed to warm to rt and stir 3h. The reaction was dumped into a separatory funnel containing saturated NH4C1, extracted with 2 x EtOAc, washed with brine, dried over Na2SO , and concentrated. The residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide l^i as a colorless oil. Data for 7^3: 'HNMR (500 MHz, CDC13) δ 7.7 (m, IH), 7.25 (m, IH), 7.1 (m, IH), 3.7 (m, 2H), 2.6 (m, 2H), 1.8 (m, 2H), 0.9 (s, 9H), 0.1 (s, 6H) ppm.
Step2 3-[3-(2,5-difluorophenyl)-l-(moφholin-4-ylcarbonyl)-5-phenyl-4,5- dihvdro-lH-pyrazol-5-yllpropan-l-ol (7-6)
To a suspension of 3.7g (18 mmol) of CuBr-DMS in THF at -78°C was added 19 mL (36 mmol) of a 1.9M solution of PhLi in cyclohexane/ether. The reaction was slowly warmed to -30°C and held there for 30 min. After recooling to - 78°C, 7^3 in THF was added and stirring was continued for 3h at that temperature. The reaction was quenched with saturated NH CI. Following extraction with 2 x EtOAc, the organic phases were combined, washed with brine, dried over Na2SO , and concentrated. The residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide an (E)/(Z) mixture of 74 as a yellow oil. To 525mg (1.3 mmol) of 74 in CH C12 was added 70 μL (1.4 mmol) of hydrazine hydrate and the mixture was heated to 140°C in a microwave reactor for 10 min. The reaction was diluted with CH C12, dried over Na SO , and concentrated. The residue was dissolved in THF, 350 μL (2.5 mmol) of triethylamine and 187 mg (0.63 mmol) of triphosgene were added, and the mixture was stirred rapidly for 6h. Moφholine (1.1 mL, 12.6 mmol) was added, stirring was continued for 30 min, and the mixture was dumped into a separatory funnel with 1M HC1. After extraction with 2 x EtOAc, the organic phases were combined, washed with brine, dried over Na2SO4, and concentrated. The residue was dissolved in dry CH3CN, 3 mL of HF-TEA was added, and the reaction was allowed to stir overnight. Following a quench with saturated NaHCO3, the mixture was extracted with 3 x EtOAc, washed with brine, dried over Na2SO , and concentrated. The residue was purified by column chromatography on silica gel with CHCl3/EtOAc/MeOH to provide 7^6 as a white solid. Data for 7^6: 'HNMR (500 MHz, CDC13) δ 7.5 (m, IH), 7.4-7.2 (m, 5H), 7.1- 7.0 (m, 2H), 3.8-3.4 (m, 13H), 2.9 (m, IH), 2.3 (m, IH), 1.7 -1.6 (m, 3H) ppm.
Step 3: 3-[3-(2,5-difluorophenyl)-l-(moφholin-4-ylcarbonyl)-5-phenyl-4,5- dihvdro-lH-pyrazol-5-yllpropan-l-amine (7-7) To 268mg (0.62 mmol) of 1^6 in CH2C12 was added 175 μL (1.24 mmol) of triethylamine and 75 μL (0.93 mmol) of mesyl chloride. After stirring for lh, the mixture was dumped into a separatory funnel with saturated NaHCO3, extracted twice with CH2C12, washed with water, and dried over Na2SO4. The residue was dissolved in DMF, 82mg (1.24 mmol) of sodium azide was added, and the reaction was heated for 4h at 65°C. After cooling to rt, the mixture was dumped into brine, extracted twice with EtOAc, and then washed with brine. The organic layer was dried over Na2SO4, concentrated, redissolved in THF and 205mg (0.78 mmol) of triphenylphosphine was added. After stirring for 15 h, 1 mL of water was added and the mixture was heated at 65°C for lh. The reaction was worked up with EtOAc and brine, dried over Na2SO , concentrated, and purified by column chromatography on silica gel with CH2Cl2/MeOH/TEA to provide H as a white solid. Data for 7-7: 1HNMR (500 MHz, CDC13) δ 7.5 (m, IH), 7.4-7.2 (m, 5H), 7.1-7.0 (m, 2H), 3.8-3.6 (m, 4H), 3.6 (m, 4H), 3.5 (m, IH), 3.4 (m, IH), 2.9 -2.8 (m, 3H), 2.2 (m, IH), 1.6 (m, 2H) ppm. HRMS (ES) calc'd M + Na for C23H26F2N4O2: 429.2097. Found: 429.2097.
The compounds of the invention illustrated below in Tables 3 and 4 were prepared by the synthetic methods described hereinabove, but substituting the appropriate acetophenones, phenyl aldehydes, carboxylic acids and amines for the corresponding reagents utilized in the above examples:
TABLE 3
Figure imgf000089_0001
R R, HRMS (calc'd) HRMS (found)
Me Me 315.1304 315.1307
NHMe Me 330.1413 330.1413
NMe2 Me 344.1569 344.1570
NHMe Et 344.1569 344.1562
NHMe CH2OH 346.1362 346.1358
— N Me 370.1726 370.1725
-N Me 368.1569 368.1564
TABLE 4
Figure imgf000090_0001
R sub
R1 R5 HRMS (calc'd) HRMS (found)
Me Me OH 303.1307 303.1304
CH2C(0)OEt Me H 359.1566 359.1560
CH2C(0)OEt H H 345.1415 345.1417
CH(CH3)C(0)OEt Me H 359.1566 359.1560
SCHEME 8
,
Figure imgf000091_0001
5-1
Figure imgf000091_0002
3-(2,5-difluorophenyl)-l-(methylsulfonyl)-5-phenyl-4,5-dihydro-lH-pyrazole (8-1) A solution of 410mg (1.7 mmol) of chalcone 54 in CH2C1 and 125 μL (2.5 mmol) of hydrazine hydrate was heated in a microwave reactor for 5 min at 150°C. To this mixture was added a catalytic amount of DMAP, lmL of triethylamine, and 1.5g (8.5 mmol) of methanesulfonic anhydride, and the reaction was stirred overnight at room temperature. The mixture was then dumped into water, extracted twice with CH2C12, washed with IM HCl, NaHCO3, and water, and dried over Na2SO4. After concentration, the residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide 84 as a white solid. Data for 84: JHNMR (500 MHz, CDC13) δ 7.7 (m, IH), 7.4-7.3 (m, 5H), 7.1 (m, 2H), 5.4 (m, IH), 3.9 (m, IH), 3.4 (m, IH), 3.0 (s, 3H) ppm. HRMS (ES) calc'd M + H for C16H14F2N2O2S1: 359.0636. Found: 359.0642. SCHEME 9
1. Dess-Martin Periodinane
2. Na(OAc)3, Me2NH-HCl, TEA, 1,2-dichloroethane
Figure imgf000092_0001
3-(2,5-difluorophenyl)-5-r3-(dimethylamino)propyl1-N-ethyl-5-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide (9-2)
To a rapidly stirring solution of 500mg (1.25 mmol) of alcohol 94 in CH2C12 was added 636mg (1.5 mmol) of Dess-Martin Periodinane (Lancaster Synthesis) and the mixture was allowed to stir for lh. A portion of 5% aqueous Na2SO3 was added, followed by saturated NaHCO3, and the biphasic mixture was stirred rapidly for lh. The mixture was then dumped into a separatory funnel, the layers were separated, and the organic layer was washed again with NaHCO3, then water, dried over Na2SO4, and concentrated to a provide the aldehyde as a white solid. To 28mg (0.07 mmol) of this aldehyde in 1,2-dichloroethane was added 6 mg (0.07 mmol) of dimethylamine hydrochloride, 10 μL (0.07 mmol) of triethylamine and 15mg (0.07 mmol) of sodium triacetoxyborohydride. After stirring overnight at room temperature, the reaction was diluted with EtOAc, washed with NaHCO3 and brine, dried over MgSO4, and concentrated. The residue was then purified by reverse phase HPLC to provide 9z2 as a colorless oil. Data for 9X2: 1HNMR (500 MHz, CDC13) δ 7.6 (m, IH), 7.4-7.2 (m, 5H), 7.1 (m, 2H), 6.1 (m, IH), 3.6-3.3 (m, 5H), 2.8 (m, IH), 2.3 (m, IH), 2.25 (s, 6H), 2.2 (m, IH), 1.6-1.4 (m, 2H) 1.2 (m, 3H) ppm. HRMS (ES) calc'd M + H for C24H30F2N4O: 429.2461. Found: 429.2440. SCHEME 10
Figure imgf000093_0001
Figure imgf000093_0002
3-(2,5-difluorophenyl)-N-ethyl-5-|3-f(lH-imidazol-2-ylcarbonyl)amino]propyl|-5- phenyl-4,5-dihvdro-lH-pyrazole-l-carboxamide (10-2)
A DMF solution was made by mixing 1 mol equivalent of each of the following: amine 10-1, imidazole-2-carboxylic acid, triethylamine, HO AT, and EDCI. The resultant mixture was stirred overnight, and then loaded directly onto a reverse phase HPLC for purification, providing 10^ as a white solid. Data for 10^2: 1HNMR (500 MHz, CDC13) δ 8.8 (bs, IH), 7.8 (m, IH), 7.6 (m, IH), 7.3-7.2 (m, 5H), 7.1 (s, 2H), 7.0 (m, 2H), 6.2 (m, IH), 3.6-3.3 (m, 6H), 2.95 (m, IH), 2.2 (m, IH), 1.6 (m, 2H), 1.2 (m, 3H) ppm. HRMS (ES) calc'd M + H for C25H26F2N6O2: 481.2158. Found: 481.2189.
The compounds of the invention illustrated below in Tables 5 and 5a were prepared by the synthetic methods described hereinabove, but substituting the appropriate acetophenones, phenyl aldehydes, carboxylic acids and amines for the corresponding reagents utilized in the above examples. Unless otherwise indicated, the compound was isolated as the free base: TABLE 5
Figure imgf000094_0001
(calc'd) HRMS (found)
NHMe F 0 NH2 359.1678 359.1654
NHEt F 1 NH2 409.1810 409.1842
NHEt F 2 NH2 401.2147 401.2160
NHEt F 1 NHC(0)Ph 491.2253 491.2247
NHEt F 2 NMe2 429.2461 429.2440
NHEt F 2 NMe2(0) 445.2410 445.2387
NHEt F 2 NHCH24-py 492.2570 492.2569
(isolated as the bis-TFA salt)
NHEt F 2 NHBn 491.2617 491.2582
TABLE 5a
R
Figure imgf000095_0001
calc'd) HRMS (found)
OH 400.1831 400.1833
-N
NH 399.1991 399.1974
Cl 1 NH2 427.1696 427.1694
-N (isolated as the HCl salt)
Cl 1 OH 428.1536 428.1536
F 1 NHC(NH)NH2 471.2315 471.2319
Figure imgf000095_0002
(isolated as the TFA salt)
F 1 NH2 429.2097 429.2097
SCHEME 11
1. Dess-Martin Periodinane
2. PhMgBr, THF
Figure imgf000096_0001
Figure imgf000096_0002
3-(2,5-difluorophenyl)-5-(3-hvdroxy-3-phenylpropyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-lH-pyrazole-1-carboxamide (11-1)
To a rapidly stirring solution of 300mg (0.78 mmol) of alcohol 94 in CH2C12 was added 395mg (0.93 mmol) of Dess-Martin Periodinane (Lancaster Synthesis) and the mixture was allowed to stir for lh. A portion of 5% aqueous Na SO3 was added, followed by saturated NaHCO3, and the biphasic mixture was stirred rapidly for lh. The mixture was then dumped into a separatory funnel, the layers were separated, and the organic layer was washed again with NaHCO3, then water, dried over Na2SO4, and concentrated to a provide the aldehyde as a white solid. To 150mg (0.039 mmol) of this aldehyde in THF at -78°C was added 260 μL (0.78 mmol) of a 3M solution of PhMgBr in Et2O. After stirring for 2h, the reaction was quenched with saturated NHtCl, dumped into a separatory funnel with EtOAc, the phases were separated, and the organic phased was washed with brine, dried over Na2SO , and concentrated. The residue was then purified by silica gel chromatography with EtOAc/hexanes to provide 11-1 as a white solid. Data for 11-1: HRMS (ES) calc'd M + H for C27H27F2N3O2: 464.2144. Found: 464.2142.
5-(3-amino-3-phenylpropyl)-3-(2,5-difluorophenyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-lH-pyrazole-l-carboxamide (11-2)
To 50mg (0.11 mmol) of 144 in CH2C12 was added 40 μL (0.28 mmol) of triethylamine and 13 μL (0.16 mmol) of mesyl chloride. After stirring for lh, the mixture was dumped into a separatory funnel with saturated NaHCO3, extract twice with CH2C12, washed with water, and dried over Na2SO4. The residue was dissolved in DMF, 13mg (0.22 mmol) of sodium azide was added, and the reaction was heated in a microwave reactor at 125°C for 15 min. After cooling to rt, the mixture was dumped into brine, extracted twice with EtOAc, and then washed with brine. The organic layer was dried over Na2SO4, concentrated, and the residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide the pure azide, which was dissolved in THF. To this was added 56mg (0.22 mmol) of triphenylphosphine and the reaction was heated to 45°C for 4h, at which time 1 mL of water was added and the mixture was kept at 45°C for lh more. The reaction was worked up with EtOAc and brine, dried over Na2SO4, concentrated, and purified by column chromatography on silica gel with CH2Cl2/MeOH/TEA to provide 11-2 as a colorless oil. Data for l lz2: HRMS (ES) calc'd M + Na for C27H28F2N4O: 463.2304. Found: 463.2297.
The compounds of the invention illustrated below in Table 6 were prepared by the synthetic methods described hereinabove, but substituting the appropriate acetophenones, phenyl aldehydes, carboxylic acids and amines for the corresponding reagents utilized in the above examples. The compounds in the table were isolated as the free base.
TABLE 6
Figure imgf000098_0001
R HRMS (calc'd) HRMS (found)
Me 427.2304 427.2305
CF, 481 .2022 481.2010
SCHEME 12
Figure imgf000099_0001
Figure imgf000099_0002
l-acetyl-3-(2,5-difluorophenyl)-4-methyl-5-phenyl-4,5-dihydro-lH-pyrazole (12-2) A solution of 950mg (3.7 mmol) of chalcone 12-1 in HOAc and 900 μL (19 mmol) of hydrazine hydrate was heated at 120°C for 24h. After cooling to room temperature, most of the solvent was removed by rotary evaporation, and the remaining material was partitioned between EtOAc and NaHCO3. The layers were separated, the organic was washed with NaHCO3, brine and dried over Na2SO . After concentration, the residue was purified by column chromatography on silica gel with EtOAc/hexanes to provide 12-2 as a single diastereomer of trans configuration as indicated by NOE determination. Data for 12^2: 'HNMR (500 MHz, CDC13) δ 7.7 (m, IH), 7.4-7.1 (m, 7H), 5.2 (m, IH), 3.6 (m, IH), 2.5 (s, 3H), 14 (m, 2H) ppm.
The following compounds are prepared by using the procedures described above but utilizing the appropriately substituted reagents:
Figure imgf000100_0001
R' R3 R° R1
2,5-dichlorophenyl H Ph NMe2
2-fluoro-5-cyanophenyl H Ph NMe2
2-fluoro-5-bromophenyl H Ph NMe2
;-fluoro-5-hydroxymethylphenyl H Ph NMe2
2-fluoro-5-chlorophenyl H Ph NMe2
2-fluoro-5-nitrophenyl H Ph NMe2
4-pyridyl H Ph NMe2
3-pyridyl H Ph NMe2
2-pyridyl H Ph NMe2
isopropyl H Ph NMe2
tert-butyl H Ph NMe2 cyclopropyl H Ph NMe2
isobutyl H Ph NMe2
1- imidazolyl H Ph NMe2
2-imidazolyl H Ph NMe2
2- thiazolyl H Ph NMe2
Figure imgf000101_0001
R2 R5 R6 R1'
2-oxazolyl H Ph NMe2
3-isoxazolyl H Ph NMe2
2-furanyl H Ph NMe2
3-furanyl H Ph NMe2 ,5-difluorophenyl H . 3-hydroxyphenyl NMe2
,5-difluorophenyl H 4-hydroxyphenyl NMe2
,5-difluorophenyl H 3-aminophenyl NMe2
,5-difluorophenyl H 3-(acetylamino)phenyl NMe2
,5-difluorophenyl H 3-carboxyphenyl NMe2
,5-difluorophenyl H 3 -tetrazolylpheny 1 NMe2
,5-difluorophenyl H 4-pyridyl NMe2
,5-difluorophenyl H 3 -pyridyl NMe2 ,5-difluorophenyl H 2-pyridyl NMe2
,5-difluorophenyl H 2-pyrimidinyl NMe2
,5-difluorophenyl H 6-indolyl NMe2
,5-difluorophenyl H 4-indolyl NMe2
,5-difluorophenyl H 6-benzimidazolyl NMe2
,5-difluoropheny H 1 -imidazolyl NMe2
Figure imgf000102_0001
R^ R3 R° R1 ,5-difluorophenyl H 2-imidazolyl NMe2 ,5-difluorophenyl H 2- thiazolyl NMe2 ,5-difluorophenyl H 2-oxazolyl NMe2 ,5-difluorophenyl JJ 3-isoxazolyl NMe2
,5-difluorophenyl H 2-furanyl NMe2
,5-difluorophenyl H 3 -furanyl NMe2
2,5-difluorophenyl H Ph
Figure imgf000102_0002
2,5-difluorophenyl H Ph
NH2
2,5-difluorophenyl H Ph
Figure imgf000102_0003
2,5-difluorophenyl H Ph
2,5-difluorophenyl H Ph
Figure imgf000102_0004
Figure imgf000103_0001
R2 R R6 R1'
, 5 -di fl uoroph eny 1 H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000103_0002
Figure imgf000104_0001
R R3 R° R1
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
2,5-difluorophenyl H Ph
Figure imgf000104_0002
Figure imgf000105_0001
R R3 R° R1
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000105_0002
Me ,5-difluorophenyl H Ph
N
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000105_0003
Figure imgf000106_0001
R2 jSub R6 R1'
, 5 -difl uoropheny 1 NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH, Ph
,5 -difl uoropheny 1 NH, Ph
Figure imgf000106_0002
Figure imgf000107_0001
R^ R sub R° R1
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH, Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000107_0002
Figure imgf000108_0001
, 5 -difluoropheny 1 NH2 Ph
NH2
,5-difluorophenyl NH2 Ph
Figure imgf000108_0002
,5-difluorophenyl NH2 h
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000108_0003
Figure imgf000109_0001
Rz R sub R° R1
,5-difluorophenyl NH2 Ph
Figure imgf000109_0002
,5-difluorophen
Figure imgf000109_0003
,5-difluorophenyl NH2 P
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000109_0004
N'
/
Figure imgf000110_0001
R sub R° R1
,5-difluorophenyl NH2 3-hydroxyphenyl NMe2
,5-difluorophenyl NH2 4-hydroxyphenyl NMe2
,5-difluorophenyl NH2 3-aminophenyl NMe2
,5-difluorophenyl NH2 3-(aCetylamino)phenyl NMe2
,5-difluorophenyl NH2 3-carboxyphenyl NMe2
,5-difluorophenyl NH2 3-tetrazolylρhenyl NMe2
,5-difluorophenyl NH2 4-pyridyl NMe
,5-difluorophenyl NH2 3-pyridyl NMe?
,5-difluorophenyl NH2 2-pyridyl NMβ
,5-difluorophenyl NH2 2-pyrimidinyl NMe;
Figure imgf000111_0001
R^ R sub R° R1
,5-difluorophenyl NH2 6-indolyl NMe2
,5-difluorophenyl NH2 4-indolyl NMe2
,5-difluorophenyl NH2 6-benzimidazolyl NMe2
,5-difluorophenyl NH2 x_ imidazolyl NMe,
,5-difluorophenyl NH2 2-imidazolyl NMe2
,5-difluorophenyl NH2 2- thiazolyl NMe2
,5-difluorophenyl NH2 2-oxazolyl NMe2
,5-difluorophenyl NH2 3-isoxazolyl NMe2
,5-difluorophenyl NH2 2-furanyl NMe5
,5-difluorophenyl NH2 3-furanyl NMe,
Figure imgf000112_0001
R2 sub R6 R1'
2,5-dichlorophenyl NH2 Ph NMe2
2-fluoro-5-cyanophenyl NH2 Ph NMe2
2-fluoro-5-bromophenyl NH Ph NMe2
-fluoro-5-hydroxymethylphenyl NH2 Ph NMe2
2-fluoro-5-chlorophenyl NH2 Ph NMe2
2-fluoro-5-nitrophenyl NH2 Ph NMe2
4-pyridyl H2 Ph NMe2
3-pyridyl NH, Ph NMe2
2-pyridyl NH? Ph NMe2
Figure imgf000113_0001
R^ R sub R° R1
isopropyl NH2 Ph NMe2
tert-butyl ^2 ph NMe2
cyclopropyl NH2 Ph NMe2
isobutyl NH Ph NMe5
- imidazolyl NH2 Ph NMe2
-imidazolyl NH2 Ph NMe2
- thiazolyl NH2 P NMe2
2-oxazolyl P NMe2
-isoxazolyl NH, Ph NMe5
2-furanyl NH2 Ph NMe2
3-furanyl NH2 Ph NMe2
Figure imgf000114_0001
R R sub' R° R1
,5-difluorophenyl phenyl Ph NMe2
,5-difluorophenyl 4-nitrophenyl Ph NMe;
,5-difluorophenyl 4-trifluoromethylphenyl Ph NMe2
,5-difluorophenyl 4-chlorophenyl P NMe2
,5-difluorophenyl QQ Me Ph NMe2
,5-difluorophenyl 4-pyridyl Ph NMe2
,5-difluorophenyl 3-pyridyl Ph NMe2
,5-difluorophenyl 2-pyridyl Ph NMe2
,5-difluorophenyl 2-imidazolyl Ph NMe2
,5-difluorophenyl CONH2 Ph NMe2
Figure imgf000115_0001
RΔ R sub" R° R 1'
,5-difluorophenyl phenyl Ph NMe2
,5-difluorophenyl 4-nitrophenyl Ph NMθ∑
,5-difluorophenyl 4-trifluoromethylphenyl Ph NMe2
,5-difluorophenyl 4-chlorophenyl Ph NMe2
,5-difluorophenyl CO2Me Ph NMe2
,5-difluorophenyl 4-pyridyl P NMe2
,5-difluorophenyl 3-pyridyl Ph NMe:
,5-difluorophenyl 2-pyridyl Ph NMe2
,5-difluorophenyl 2-imidazolyl Ph N e2
,5-difluorophenyl 4-cyanophenyl Ph NMe2
Figure imgf000116_0001
R2 W-R5 R6 R1'
,5-difluorophenyl . -CH2CF2CH2NH2 Ph NMe2
,5-difluorophenyl -CH2OCH2CH2NH2 Ph NMe2
,5-difluorophenyl -CH2CH2CH(CHF2)NH2 Ph NMe2
,5-difluorophenyl -CB.2S(0)2CH2Cll2Kll2 Ph NMe2
,5-difluorophenyl -CH2OCF2CH2NH2 Ph NMe2
,5-difluorophenyl -CH2CH2CF2CH2NH2 Ph NMe2
,5-difluorophenyl -CH2CH2CH2CH(CHF2)NH2 Ph NMe2
,5-difluorophenyl -CH2CH(OH)CH2CH2NH2 Ph NMe2
,5-difluorophenyl -CH2CH(OH)CH2NH2 Ph NMe2
,5-difluorophenyl -CH2C(O)CH2CH2NH2 Ph NMe2

Claims

WHAT IS CLAIMED IS:
1. A compound of Formula I:
Figure imgf000117_0001
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
a is O or l; b is O or l; m is 0, 1, or 2; n is 0 to 2; u is 1, 2, 3, 4 or 5;
Rl is selected from:
I) (C=X)Cι-Cio alkyl, 2) (C=X)aryl,
3)
Figure imgf000117_0002
alkenyl,
4) (C=X)C2-Cio alkynyl,
5) (C=X)C3-C8 cycloalkyl,
6) (C=X)heterocyclyl, 7) (C=X)NR7R8,
8) (C=X)OCι-Cio alkyl,
9) SO2NR7R8,
10) SO2Ci-Cιo alkyl,
II) SO2Ci-Cio aryl, 12) SO2C1-C10 heterocyclyl,
13) C1-C10 alkyl,
14) aryl,
15) heteroaryl,
16) (CH2)u(C=O)Cι-Cio alkyl, 17) (CH2)u(C=O) NR7R8,
18) 3-pyrrolidinonyl, 3-piperidinonyl, 2-cyclopentanonyl, 2- cyclohexanonyl,
19) (C=O)(C=O)Cι-Cιo alkyl, 20) (C=O)(C=O)NR7R8,
21) (C=O)(C=O)O Ci-Cio alkyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from:
1) Ci-Cio alkyl,
2) aryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl, 5) C1-C6 perfluoroalkyl,
6) C1-C6 aralkyl,
7) C1-C6 heteroaralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R4, R5 and R6 are independently selected from: 1) H, 2) C1-C10 alkyl,
3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl,
6) C1-C6 perfluoroalkyl, 7) C1-C6 aralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or R3 and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(O)m, -N(R9)C(O)-, and -N(COR10)-;
5 R7 is
1) (C=O)aObCι-Cιo alkyl,
2) (C=O)aObaryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl,
10 5) (C=O)aOb heterocyclyl,
6) CO2H,
7) halo,
8) CN,
9) OH,
15 10) O Cι-C6 perfluoroalkyl,
Figure imgf000119_0001
12) S(O)mRa,
Figure imgf000119_0002
14) oxo,
20 15) CHO,
16) (N=O)R9RlO, or
17) (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R8; ηc
ZJ
Rδ is ; selected from:
1)
Figure imgf000119_0003
wherein r and s are independently 0 or 1,
2) Or(Cι-C3)perfluoroalkyl, wherein r is 0 or 1,
3) (Co-C6)alkylene-S(O)mRa, wherein m is 0, 1, or 2,
30 4) oxo,
5) OH,
6) halo,
7) CN,
8) (C=O)rOs(C2-Cio)alkenyl, 9) (C=O)rOs(C2-Clθ)alkynyl,
10) (C=O)rOs(C3-C6)cycloalkyl,
11) (C=O)rOs(Co-C6)alkylene-aryl,
12) (C=O)rOs(Co-C6)alkylene-heterocyclyl, 13) (C=O)rOs(Co-C6)alkylene-N(Rb)2,
14) C(O)Ra,
15) (Co-C6)alkylene-CO2Ra,
16) C(O)H,
17) (Cθ-C6)alkylene-CO2H, 18) C(O)N(Rb)2,
19) S(O)mRa,
Figure imgf000120_0001
21) C(NH)NH2; said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from:
I) H, 2)
Figure imgf000120_0002
alkyl,
3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl,
6) C1-C10 alkyl, 7) aryl,
8) C2-C10 alkenyl,
9) C2-C10 alkynyl,
10) heterocyclyl,
II) C3-C8 cycloalkyl, 12) SO2Ra, and
13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R8, or R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 3-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
Ra is (Cι-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Cι-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCι-C6 alkyl, (C=O)Cι-C6 alkyl or S(O)2Ra;
X is selected from O and S;
Y, W and Z are independently selected from: a bond, C=O, C=S, S(O)n, CH(OH) and O.
2. ■ The compound according to Claim I of the Formula I:
Figure imgf000121_0001
I
or a pharmaceutically acceptable salt or stereoisomer thereof, wherein
a is O or l; b is O or l; m is 0, 1, or 2; n is 0 to 2; u is 2, 3, 4 or 5;
Rl is selected from:
1) (C=X)Ci-Cιo alkyl,
2) (C=X)aryl, 3) (C=X)C2-Ciθ alkenyl,
4) (C=X)C2-C 10 alkynyl,
5) (C=X)C3-C8 cycloalkyl,
6) (C=X)heterocyclyl,
7) (C=X)NR7R8,
8) (C=X)OCι-Cιo alkyl,
9) SO2NR7R8,
10) SO Cι-Cιo alkyl,
11) SO2Ci-Cio aryl,
12) SO2Cι-Cιo heterocyclyl,
13) Ci-C 10 alkyl,
14) aryl,
15) heteroaryl,
16)
Figure imgf000122_0001
alkyl,
17) (CH2)u(C=O) NR7R8,
18) 3-pyrrolidinonyl, 3-piperidinonyl, 2-cyclopentanonyl. 2- cyclohexanonyl,
19) (C=O)(C=O)Cι-Cιo alkyl,
20) (C=O)(C=O)NR7R8,
21) (C=O)(C=O)O Ci-Cio alkyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, heteroaryl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from:
1) Ci-Cio alkyl,
2) aryl,
3) C2-C10 alkenyl,
4) C-2-C 10 alkynyl,
5) C1-C6 perfluoroalkyl,
6) C1-C6 aralkyl,
7) C1-C6 heteroaralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R45 R5 and R6 are independently selected from: 1) H,
2) Ci-Cio alkyl,
3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl, 6) C1-C6 perfluoroalkyl,
7) C1-C6 aralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R3 and R4, or R5 and R6, attached to the same carbon atom (W and Z are a bond) are combined to form -(CH2)u- wherein one of the carbon atoms is optionally replaced by a moiety selected from O, S(0)m, -N(R9)C(O)-, and -N(COR10)-;
R7 IS:
1) (C=O)aObCι-Cιo alkyl,
2) (C=O)aObaryl,
3) C2-C 10 alkenyl,
4) C2-C10 alkynyl,
5) (C=O)aOb heterocyclyl,
6) CO2H,
7) halo,
8) CN,
9) OH,
10) ObCl-C6 perfluoroalkyl,
11) Oa(C=O)bNR9RlO;
12) S(O)mRa,
Figure imgf000123_0001
14) oxo,
15) CHO,
16) (N=O)R9R105 or
17) (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one or more substituents selected from R8;
R8 is selected from:
I)
Figure imgf000124_0001
wherein r and s are independently 0 or 1, 2) Or(Cι-C3)perfluoroalkyl, wherein r is 0 or 1,
3) (Co-C6)alkylene-S(O)mRa, wherein m is 0, 1, or 2,
4) oxo,
5) OH,
6) halo, 7) CN,
8) (C=O)rOs(C2-Cio)alkenyl,
Figure imgf000124_0002
10) (C=O)rOs(C3-C6)cycloalkyl,
I I) (C=O)rOs(Co-C6)alkylene-aryl, 12) (C=O)rOs(Co-C6)alkylene-heterocyclyl,
13) (C=O)rOs(Co-C6)alkylene-N(Rb)2,
14) C(O)Ra,
15) (Co-C6)alkylene-CO2Ra,
16) C(O)H, 17) (Co-C6)alkylene-CO2H
18) C(O)N(Rb)2,
19) S(O)mRa, and
Figure imgf000124_0003
said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from: 1) H, 2) (C=O)ObCι-Cιo alkyl,
3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl, 6) Ci-Cio alkyl,
7) aryl,
8) C2-C ιo alkenyl,
9) C2-C10 alkynyl,
10) heterocyclyl, 11) C3-C8 cycloalkyl,
12) SO2Ra and
13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one or more substituents selected from R8, or
R9 and R can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic or bicyclic heterocycle optionally substituted with one or more substituents selected from R8;
Ra is (Cι-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Cι-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCι-C6 alkyl, (C=O)Ci-C6 alkyl or S(O)2Ra;
X is selected from O and S;
Y, W and Z are independently selected from: a bond, C=O, C=S, S(O)n, CH(OH) and O.
3. A compound of the Formula II,
Figure imgf000126_0001
wherein:
a is 0 or 1 ; b is O or l; m is 0, 1, or 2; n is 0 to 2;
Rl is selected from: 1) (C=O)Cι-Cιo alkyl,
2) (C=O)aryl,
3) (C=O)C2-Cιo alkenyl,
4)
Figure imgf000126_0002
alkynyl,
5) (C=O)C3-C8 cycloalkyl, 6) (C=O)heterocyclyl,
7) (C=O)OCι-Cιo alkyl,
8) (C=O)NR7R8,
9) SO2NR7R8,
10) SO2Ci-Cιo alkyl, 11) SO2Cι-Cιo aryl,
12) SO2Cι-Cιo heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl is optionally substituted with one or more substituents selected from R7; or
R2 is selected from:
1) Ci-Cio alkyl,
2) aryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl, 5) C1-C6 perfluoroalkyl,
6) C1-C6 aralkyl,
7) C3-C8 cycloalkyl, and
8) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R3, R4, R5 and R6 are independently selected from:
1) H, 2) C1-C10 alkyl,
3) aryl,
4) C2-C10 alkenyl,
5) C2-C10 alkynyl,
6) C1-C6 perfluoroalkyl, 7) C1-C6 aralkyl,
8) C3-C8 cycloalkyl, and
9) heterocyclyl, said alkyl, aryl, alkenyl, alkynyl, cycloalkyl, aralkyl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
R7 IS:
1) (C=O)aObCι-Cιo alkyl,
2) (C=O)aObaryl,
3) C2-C10 alkenyl,
4) C2-C10 alkynyl,
5) (C=O)aOb heterocyclyl,
6) CO2H,
7) halo,
8) CN,
9) OH,
10) ObCι-C6 perfluoroalkyl,
Figure imgf000127_0001
12) S(O)mRa,
Figure imgf000127_0002
14) oxo,
15) CHO,
16) (N=O)R9R10, or
17) (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl optionally substituted with one, two or three substituents selected from R8;
R8 is selected from:
1)
Figure imgf000128_0001
wherein r and s are independently 0 or 1,
2) Or(Cι-C3)perfluoroalkyl, wherein r is 0 or 1,
3) oxo,
4) OH,
5) halo,
6) CN,
7) (C2-Cio)alkenyl,
8) (C2-Cio)alkynyl,
9) (C=O)rOs(C3-C6)cycloalkyl,
10) (C=O)rOs(Co-C6)alkylene-aryl,
11) (C=O)rOs(Co-C6)alkylene-heterocyclyl,
12) (C=O)rOs(Co-C6)alkylene-N(Rb)2,
13) C(O)Ra,
14) (Co-C6)alkylene-CO2Ra,
15) C(O)H,
16) (Co-C6)alkylene-CO2H, and
17) C(O)N(Rb)2,
18) S(O)mRa, and
Figure imgf000128_0002
said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up to three substituents selected from Rb, OH, (Cι-C6)alkoxy, halogen, CO2H, CN, O(C=O)Cι-C6 alkyl, oxo, and N(Rb)2;
R9 and RlO are independently selected from: 1) H, 2)
Figure imgf000129_0001
alkyl,
3) (C=O)ObC3-C8 cycloalkyl,
4) (C=O)Obaryl,
5) (C=O)Obheterocyclyl, 6) Ci-Cio alkyl,
7) aryl,
8) C2-C10 alkenyl,
9) C2-C10 alkynyl,
10) heterocyclyl, 11) C3-C8 cycloalkyl,
12) SO2Ra, and
13) (C=O)NRb2, said alkyl, cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted with one, two or three substituents selected from R8, or
R9 and RlO can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle with 5-7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocylcic or bicyclic heterocycle optionally substituted with one, two or three substituents selected from R8;
Ra is (Cι-C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is H, (Cι-C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl, (C=O)OCι-C6 alkyl, (C=O)C 1 -Cβ alkyl or S (O)2Ra
4. The compound according to Claim 3 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:
Rl is selected from:
1) (C=O)Cι-Cιo alkyl,
2) (C=O)aryl,
3) (C=O)C3-C8 cycloalkyl,
4) (C=O)heterocyclyl, 5) (C=O)OCι-Cιo alkyl,
6) SO2NR7R8, and
7) SO2Cl-Cio alkyl, said alkyl, aryl, cycloalkyl, and heterocyclyl is optionally substituted with one, two or three substituents selected from R7;
R2 is selected from:
1) Ci-Cio alkyl,
2) aryl, and 3) heteroaryl, said alkyl, aryl and heteroaryl is optionally substituted with one or more substituents selected from R7;
R3 and R are independently selected from: 1) H, and
2) Ci-Cio alkyl, said alkyl is optionally substituted with one or more substituents selected from R7; and
R5 and R6 are independently selected from:
1) H,
2) Ci-C 10 alkyl,
3) aryl, and
4) heterocyclyl, said alkyl, aryl and heterocyclyl is optionally substituted with one or more substituents selected from R7;
and R7, R8, R9, R10, Ra and Rb are as described in Claim 2.
5. The compound according to Claim 4, or the pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is phenyl, optionally substituted with one or two substittuents selected from R7.
6. A compound selected from: l-acetyl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3-(2-chlorophenyl)-l-isobutyryl-4,5-dihydro-lH-pyrazol-5-yl]phenol
341-acetyl-3-(2-chlorophenyl)-5-methyl-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-acetyl-3-(2,5-difluorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3- l-Acetyl-3-(2-fluorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Acetyl-3-(3-bromophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Acetyl-3-(2,3-dichlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3-[l-Acetyl-3-(2,5-dichlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Propionyl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
3-[l-Isobutyryl-3-(2-chlorophenyl)-4,5-dihydro-lH-pyrazol-5-yl]phenol
l-Acetyl-3-(2-chlorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(3-chlorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-Acetyl-3-(4-fluoro-3-hydroxyphenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
l-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro4H-pyrazol-l- yl]carbonyl } piperazine
3-(2,5-difluorophenyl)- N,N-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide 3-(2,5-difluorophenyl)-5-(3-hydroxyphenyl)-N,N-dimethyl-4,5-dihydro-lH-pyrazole- 1 -carboxamide
4-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl} moφholine
3-[3-(2,5-difluorophenyl)-l-(morpholin-4-ylcarbonyl)-4,5-dihydro-lH-pyrazol-5- yljphenol
3-(2,5-difluorophenyl)- N,N-diethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole-l-carboxamide
3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH-pyrazole
3-[3-(2-fluoro-5-methylphenyl)- 1 -(pyrrolidin- 1 -ylcarbonyl)-4,5-dihydro- lH-pyrazol- 5-yl]phenol
l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazole
3-[l-(azetidin-l-ylcarbonyl)-3-(5-chloro-2-fluorophenyl)-4,5-dihydro-lH-pyrazol-5- yljphenol
l-(l-{ [3-(5-chloro-2-fluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl}piperidin-2-yl)-N,N-dimethylmethanamine
3-(2,5-difluorophenyl)-l,5-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole
l-acetyl-3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-N,5-dimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide 3-(2,5-difluorophenyl)-N,N,5-trimethyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-ethyl-N-methyl-5-phenyl-4,5-dihydro-lH-pyrazole-l- carboxamide
3-(2,5-difluorophenyl)-5-(hydroxymethyl)-N-methyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
3-(2,5-difluorophenyl)-5-methyl-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro- 1 H-pyrazole
3-(2,5-difluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-methyl-5-phenyl- 4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-l,5-dimethyl-5-(3-hydroxyphenyl)-4,5-dihydro-lH-pyrazole
ethyl [3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazol-l-yl]acetate
ethyl [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l-yl]acetate
ethyl 2-[3-(2,5-difluorophenyl)-5-methyl-5-phenyl-4,5-dihydro-lH-pyrazol-l- yljpropanoate
3-[3-(2,5-difluorophenyl)-l-(morpholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l -amine 3-(2,5-difluorophenyl)-l-(methylsulfonyl)-5-phenyl-4,5-dihydro-lH-pyrazole
3-(2,5-difluorophenyl)-5-[3-(dimethylamino)propyl]-N-ethyl-5-phenyl-4,5-dihydro- lH-pyrazole-1-carboxamide
3-(2,5-difluorophenyl)-N-ethyl-5-{3-[(lH-imidazol-2-ylcarbonyl)amino]propyl}-5- phenyl-4,5-dihydro-lH-pyrazole-l-carboxamide 5-(2-aminoethyl)-3-(2,5-difluorophenyl)-N-methyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
5-(3-aminopropyl)-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
5-(3-aminobutyl)-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH-pyrazole- 1 -carboxamide
5-[3-(benzoylamino)propyl]-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro- lH-pyrazole-1 -carboxamide
3-(2,5-difluorophenyl)-5-[4-(dimethylamino)butyl]-N-ethyl-5-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide
3-(2,5-difluorophenyl)-5-[4-(dimethylnitroryl)but-l-yl]-N-ethyl-5-phenyl-4,5- dihydro-lH-pyrazole-1-carboxamide
5-[4-(benzylamino)butyl]-3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-4,5-dihydro-lH- pyrazole-1 -carboxamide
3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-5-{4-[(pyridin-4-ylmethyl)amino]butyl}- 4,5-dihydro-lH-pyrazole-l-carboxamide
3-[l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l-ol
3-[l-(azetidin-l-ylcarbonyl)-3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propan-l-amine
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro- 1 H-pyrazol-5-yl]propan- 1 -ol 3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l-amine
3-[3-(5-chloro-2-fluorophenyl)-l-(morpholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro- lH-pyrazol-5-yl]propan-l-amine
N-{3-[3-(2,5-difluorophenyl)-l-(morpholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propyl}guanidine
5-(3-amino-3-phenylpropyl)-3-(2,5-difluorophenyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-lH-pyrazole-1-carboxamide
3-[3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH- pyrazol-5-yl]-l-methylpropylamine
3-[3-(2,5-difluorophenyl)-5-phenyl-l-(pyrrolidin-l-ylcarbonyl)-4,5-dihydro-lH- pyrazol-5-yl]-l-(trifluoromethyl)propylamine
l-acetyl-3-(2,5-difluorophenyl)4-methyl-5-phenyl-4,5-dihydro-lH-pyrazole
or a pharmaceutically acceptable salt or stereoisomer thereof.
7. A compound selected from: l-{ [3-(2,5-difluorophenyl)-5-phenyl-4,5-dihydro-lH-pyrazol-l- yl]carbonyl}piperazine TFA salt,
3-(2,5-difluorophenyl)-N-ethyl-5-phenyl-5-{4-[(pyridin-4-ylmethyl)amino]butyl}- 4,5-dihydro-lH-pyrazole-l-carboxamide bis TFA salt,
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l-amine hydrochloride salt, and
N-{3-[3-(2,5-difluorophenyl)-l-(morpholin-4-ylcarbonyl)-5-phenyl-4,5-dihydro-lH- pyrazol-5-yl]propyl}guanidine TFA salt.
8. The compound according to Claim 6 which is selected from:
3-[l-acetyl-3-(2,5-difluorophenyl)4,5-dihydro-lH-pyrazol-5-yl]phenol
Figure imgf000136_0001
3-(2,5-difluorophenyl)-5-(3-hydroxy-3-phenylpropyl)-N,N-dimethyl-5-phenyl-4,5- dihydro-lH-pyrazole-1-carboxamide
Figure imgf000136_0002
3-[3-(5-chloro-2-fluorophenyl)-l-(2,5-dihydro-lH-pyrrol-l-ylcarbonyl)-5-phenyl-4,5- dihydro-lH-pyrazol-5-yl]propan-l -amine
Figure imgf000136_0003
or a pharmaceutically acceptable salt or stereoisomer thereof.
9. A compound selected from:
Figure imgf000137_0001
R R= R° R1
2,5-dichlorophenyl H Ph NMe2
2-fluoro-5-cyanophenyl H Ph NMe2
2-fluoro-5-bromophenyl H Ph NMe2 -fluoro-5-hydroxymethylphenyl H Ph NMe2
2-fluoro-5-chlorophenyl H Ph NMe2
2-fluoro-5-nitrophenyl H Ph NMe2
4-pyridyl H Ph NMe2
3-pyridyl H Ph NMe2
2-pyridyl H Ph NMe2
isopropyl H Ph NMe2
tert-butyl H Ph NMe2 cyclopropyl H Ph NMe2
isobutyl H Ph NMe2
1- imidazolyl H Ph NMe2
2-imidazolyl H Ph NMe2
2- thiazolyl H Ph NMe2
Figure imgf000138_0001
R2 R5 R6 R1'
2-oxazolyl H Ph NMe2
3-isoxazolyl H Ph NMe2
2-furanyl H Ph NMe2
3-furanyl H Ph NMe2 ,5-difluorophenyl H . 3-hydroxyphenyl NMe2
,5-difluorophenyl H 4-hydroxyphenyl NMe2
, 5 -difluoropheny 1 H 3-aminophenyl NMe2
,5-difluorophenyl H 3 -(acetylamino)pheny 1 NMe2
,5-difluorophenyl H 3-carboxyphenyl NMe2
,5-difluorophenyl H 3-tetrazolylphenyl NMe2
,5-difluorophenyl H 4-pyridyl NMe2
,5-difluorophenyl H 3 -pyridyl NMe2 ,5-difluorophenyl H 2-pyridyl NMe2
,5-difluorophenyl H 2-pyrimidinyl NMe2
, 5 -difluoropheny 1 H 6-indolyl NMe2
,5-difluorophenyl H 4-indolyl NMe2
,5-difluorophenyl H 6-benzimidazolyl NMe2
,5-difluoropheny H 1 -imidazolyl NMe2
Figure imgf000139_0001
R2 R5 R6 R1 ,5-difluorophenyl H 2-imidazolyl NMe2 ,5 -difluoropheny 1 H 2- thiazolyl NMe2 , 5 -di fluorophenyl H 2-oxazolyl NMe2 ,5-difluorophenyl H 3 -isoxazolyl NMe2
,5-difluorophenyl H 2-furanyl NMe2
,5-difluorophenyl H 3 -furanyl NMe2
2,5-difluorophenyl H Ph
2,5-difluorophenyl H Ph
Figure imgf000139_0002
2,5-difluorophenyl H Ph
NH2
2,5-difluorophenyl H Ph
2,5-difluorophenyl H Ph
Figure imgf000139_0003
Figure imgf000140_0001
R^ R R° R1
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000140_0002
Figure imgf000141_0001
R" R3 R° R1
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000141_0002
Figure imgf000142_0001
R R3 R° R1
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
,5-difluorophenyl H Ph
Figure imgf000142_0002
Figure imgf000143_0001
sub
R2 R6 R1'
, 5 -difluoropheny 1 NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000143_0002
Figure imgf000144_0001
R R sub R° R1
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000144_0002
Figure imgf000145_0001
R2 sub R6 R1'
,5-difluorophenyl NH2 Ph
NH2
,5-difluorophenyl NH2 Ph /
NH 2
,5-difluorophenyl NH2 Ph i_
NH 2
,5-difluorophenyl NH2 Ph
NH2
,5-difluorophenyl NH2 Ph
Figure imgf000145_0002
Figure imgf000146_0001
R^ R sub R° R1
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
,5-difluorophenyl NH2 Ph
Figure imgf000146_0002
,5-difluorophenyl NH2 Ph HN
,5-difluorophenyl NH2 Ph
Figure imgf000146_0003
N'
/
Figure imgf000147_0001
R R! sub R° R1
,5-difluorophenyl NH2 3-hydroxyphenyl NMe2
,5-difluorophenyl NH2 4-hydroxyphenyl NMe2
,5-difluorophenyl NH2 3-aminophenyl NMe2
,5-difluorophenyl NH2 3-(acetylamino)phenyl NMe2
,5-difluorophenyl NH2 3-carboxyphenyl NMe-
,5-difluorophenyl NH2 3-tetrazolylphenyl NMe2
,5-difluorophenyl NH2 4-pyridyl NMe,
,5-difluorophenyl NH2 3-pyridyl NMβo
,5-difluorophenyl NH2 2-pyridyl NMe,
,5-difluorophenyl NH2 2-pyrimidinyl NMe,
Figure imgf000148_0001
sub
R R R° R1
,5-difluorophenyl NH2 6-indolyl NMe,
,5-difluorophenyl NH2 4-indolyl NMe,
,5-difluorophenyl NH2 6-benzimidazolyl NMe2
,5-difluorophenyl NH2 ι_ imidazolyl NMe,
,5-difluorophenyl NH2 2-imidazolyl NMe,
,5-difluorophenyl NH2 2- thiazolyl NMe,
,5-difluorophenyl NH2 2-oxazolyl NMe,
,5-difluorophenyl NH2 3-isoxazolyl NMe,
,5-difluorophenyl NH2 2-furanyl NMe,
,5-difluorophenyl NH2 3-furanyl NMe,
Figure imgf000149_0001
R2 ub R6 R1'
2,5-dichlorophenyl NH2 Ph NMe2
2-fluoro-5-cyanophenyl NH2 Ph NMe2
2-fluoro-5-bromophenyl NH2 Ph NMe2
-fluoro-5-hydroxymethylρhenyl NH2 Ph NMe2
2-fluoro-5-chlorophenyl NH2 Ph NMe2
2-fluoro-5-nitrophenyl NH2 Ph NMe2
4-pyridyl NH2 ph NMe2
3-pyridyl NH2 Ph NMe2
2-pyridyl NH2 Ph NMe2
Figure imgf000150_0001
Rz R sub R° R1
isopropyl NH2 ph NMe2
tert-butyl ^ ph NMe2
cyclopropyl NH2 Ph NMe2
isobutyl NH2 Ph NMe2
- imidazolyl NH2 Ph NMe2
-imidazolyl NH2 Ph NMe2
- thiazolyl NH2 Ph NMe2
2-oxazolyl NH9 Ph NMe2
-isoxazolyl NH, Ph NMe,
2-furanyl NH2 Ph NMe2
3-furanyl NH2 Ph NMe2
Figure imgf000151_0001
R2 sub' R6 R1'
2,5-difluorophenyl phenyl Ph NMe2
, 5 -difluoropheny 1 4-nitrophenyl Ph NMe2
,5-difluorophenyl 4-trifluoromethylphenyl Ph NMe2
,5-difluorophenyl 4-chlorophenyl Ph NMe2
,5-difluorophenyl CQ Me Ph NMe;
,5-difluorophenyl 4-pyridyl Ph NMe2
,5-difluorophenyl 3-pyridyl Ph NMe2
,5-difluorophenyl 2-pyridyl Ph NMe2
,5-difluorophenyl 2-imidazolyl Ph NMe2
,5-difluorophenyl CONH2 Ph NMe2
Figure imgf000152_0001
R R sub': R° R1'
,5-difluorophenyl phenyl Ph NMe2
,5-difluorophenyl 4-nitrophenyl h NMe2
,5-difluorophenyl 4-trifUιoromethylphenyl Ph NMe2
,5-difluorophenyl 4-chlorophenyl Ph NMe2
,5-difluorophenyl CO2Me Ph NMe2
,5-difluorophenyl 4-pyridyl Ph NMe2
,5-difluorophenyl 3-nyridyl Ph N e
,5-difluorophenyl 2-pyridyl Ph NMe2
,5-difluorophenyl 2-imidazolyl ph NMe2
,5-difluorophenyl 4-cyanophenyl Ph NMe2
Figure imgf000153_0001
R2 W-R5 R6 R1'
2,5- difluorophenyl -CH2CF2CH2NH2 Ph NMe2
2,5- -difluorophenyl -CH OCH CH NH Ph NMe2
2,5-difluorophenyl -CH2CH2CH(CHF2)NH2 Ph NMe2
2,5-difluorophenyl -CH2S(O)2CH2CH2NH2 Ph NMe2
2,5-difluorophenyl -CH2OCF2CH2NH2 Ph NMe2
2,5-difluorophenyl -CH2CH2CF2CH2NH2 Ph NMe2
2,5-difluorophenyl -CH2CH2CH2CH(CHF2)NH2 Ph NMe2
2,5-difluorophenyl -CH2CH(OH)CH2CH2NH2 Ph NMe2
2,5-difluorophenyl -CH2CH(OH)CH2NH2 Ph NMe2
2,5-difluorophenyl -CH2C(O)CH2CH2NH2 Ph NMe2
or a pharmaceutically acceptable salt or stereoisomer thereof.
10. A pharmaceutical composition that is comprised of a compound in accordance with Claim 1 and a pharmaceutically acceptable carrier.
11. A method of treating or preventing cancer in a mammal in need of such treatment that is comprised of administering to said mammal a therapeutically effective amount of a compound of Claim 1.
12. A method of treating cancer or preventing cancer in accordance with Claim 11 wherein the cancer is selected from cancers of the brain, genitourinary tract, lymphatic system, stomach, larynx and lung.
13. A method of treating or preventing cancer in accordance with Claim 11 wherein the cancer is selected from histiocytic lymphoma, lung adenocarcinoma, small cell lung cancers, pancreatic cancer, gioblastomas and breast carcinoma.
14. A process for making a pharmaceutical composition which comprises combining a compound of Claim 1 with a pharmaceutically acceptable carrier.
15. The composition of Claim 9 further comprising a second compound selected from:
1) an estrogen receptor modulator,
2) an androgen receptor modulator, 3) a retinoid receptor modulator,
4) a cytotoxic agent,
5) an antiproliferative agent,
6) a prenyl-protein transferase inhibitor,
7) an HMG-CoA reductase inhibitor, 8) an HIV protease inhibitor,
9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor, and
11) a PPAR-γ agonist,
12) a PPAR-δ agonists; 13) an inhibitor of cell proliferation and survival signaling, and
14) an agent that interfers with a cell cycle checkpoint.
16. The composition of Claim 15, wherein the second compound is an angiogenesis inhibitor selected from the group consisting of a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of fibroblast- derived growth factor, an inhibitor of platelet derived growth factor, an MMP inhibitor, an integrin blocker, interferon- , interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-(chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, and an antibody to VEGF.
17. The composition according to Claim 10 further comprising a proteosome inhibitor.
18. The composition according to Claim 10 further comprising a aurora kinase inhibitor.
19. The composition according to Claim 10 further comprising a Raf kinase inhibitor.
20. The composition according to Claim 10 further comprising a serine/threonine kinase inhibitor.
21. The composition according to Claim 10 further comprising an inhibitor of another mitotic kinesin which is not KSP.
22. The composition of Claim 16, wherein the second compound is an estrogen receptor modulator selected from tamoxifen and raloxifene.
23. A method of treating cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with radiation therapy.
24. A method of treating or preventing cancer that comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with a compound selected from:
I) an estrogen receptor modulator, 2) an androgen receptor modulator,
3) a retinoid receptor modulator,
4) a cytotoxic agent,
5) an antiproliferative agent,
6) a prenyl-protein transferase inhibitor, 7) an HMG-CoA reductase inhibitor,
8) an HIN protease inhibitor,
9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor,
II) PPAR-γ agoni sts , 12) PPAR-δ agonists,
13) an inhibitor of inherent multidrug resistance,
14) an anti-emetic agent,
15) an agent useful in the treatment of anemia,
16) an agent useful in the treatment of neutropenia, 17) an immunologic-enhancing drug,
18) an inhibitor of cell proliferation and survival signaling, and
19) an agent that interfers with a cell cycle checkpoint.
25. A method of treating cancer that comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with radiation therapy and a compound selected from:
1) an estrogen receptor modulator,
2) an androgen receptor modulator,
3) a retinoid receptor modulator, 4) a cytotoxic agent,
5) an antiproliferative agent,
6) a prenyl-protein transferase inhibitor,
7) an HMG-CoA reductase inhibitor,
8) an HTV protease inhibitor, 9) a reverse transcriptase inhibitor,
10) an angiogenesis inhibitor,
11) PPAR-γ agonists,
12) PPAR-δ agonists, 13) an inhibitor of inherent multidrug resistance,
14) an anti-emetic agent,
15) an agent useful in the treatment of anemia,
16) an agent useful in the treatment of neutropenia,
17) an immunologic-enhancing drug, 18) an inhibitor of cell proliferation and survival signaling, and
19) an agent that interfers with a cell cycle checkpoint.
26. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 and paclitaxel or trastuzumab.
27. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 and a GPflb/IIIa antagonist.
28. The method of Claim 27 wherein the GPIIb/IIIa antagonist is tirofiban.
29. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with a COX-2 inhibitor.
30. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with a proteosome inhibitor.
31. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with an aurora kinase inhibitor.
32. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with a Raf kinase inhibitor.
33. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with a serine/threonine kinase inhibitor.
34. A method of treating or preventing cancer which comprises administering a therapeutically effective amount of a compound of Claim 1 in combination with an inhibitor of a mitotic kinesin that is not KSP.
35. A method of modulating mitotic spindle formation which comprises administering a therapeutically effective amount of a compound of Claim 1.
36. A method of inhibiting the mitotic kinesin KSP which comprises administering a therapeutically effective amount of a compound of Claim 1.
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EP1492487B1 (en) 2009-11-11
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AU2003249597B2 (en) 2007-06-28
EP1492487A2 (en) 2005-01-05
US7745639B2 (en) 2010-06-29
JP4399269B2 (en) 2010-01-13
AU2003249597A1 (en) 2003-10-08
DE60329990D1 (en) 2009-12-24
US20050119484A1 (en) 2005-06-02
CA2478068C (en) 2011-02-08
ATE448207T1 (en) 2009-11-15
EP1492487A4 (en) 2006-01-11
CA2478068A1 (en) 2003-10-02
WO2003079973A3 (en) 2004-11-11

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