HK1130788A - Thiophene-carboxamides useful as inhibitors of protein kinases - Google Patents

Description

Thiophene-carboxamides useful as protein kinase inhibitors

Field of the invention

[0001] The present invention relates to compounds useful as inhibitors of protein kinases. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various disorders. The invention also relates to methods of preparing the compounds of the invention.

Background

[0002] In recent years, better understanding of the structure of enzymes and other biomolecules associated with disease has greatly facilitated the search for new therapeutic agents. One important class of enzymes that has been the subject of extensive research is protein kinases.

[0003] Protein kinases constitute a large family of structurally related enzymes responsible for controlling a variety of signal transduction processes within cells (see Hardie, G and Hanks, S.Theprotein Kinase products Book, I and II, Academic Press, San Diego, CA: 1995). Protein kinases are thought to have evolved from a common genetic gene by preserving their structure and catalytic function. Almost all kinases contain a similar 250-300 amino acid catalytic domain. Kinases can be classified into several families according to the substrates they phosphorylate (e.g. protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs that generally correspond to each kinase family have been identified (see, e.g., Hanks, s.k., Hunter, t., FASEB j.1995, 9, 576-596; Knighton et al, Science1991, 253, 407-414; Hiles et al, Cell 1992, 70, 419-429; Kunz et al, Cell 1993, 73, 585-596; Garcia-Bustos et al, EMBO J1994, 13, 2352-2361).

[0004]In general, protein kinases mediate intracellular signaling by affecting the transfer of phosphoryl groups from nucleoside triphosphates to protein acceptors involved in signaling pathways. These phosphorylation events act as molecular switches, capable of modulating or regulating the biological function of the target protein. These phosphorylation events are ultimately stimulated in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress response signals (e.g., shock, heat shock, ultraviolet radiation, bacterial endotoxins, and H₂O₂) Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), and growth factors such as granulocyte macrophage colony stimulating factor (GM-CSF) and Fibroblast Growth Factor (FGF). Extracellular stimuli can affect one or more cellular responses involving cell growth, migration, differentiation, hormone secretion, transcription factor activation, muscle contraction, carbohydrate metabolism, protein synthesis control, and cell cycle regulation.

[0005] Many diseases are associated with abnormal cellular responses triggered by the aforementioned protein kinase-mediated events. Such diseases include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, allergy and asthma, alzheimer's disease, and hormone-related diseases. Accordingly, there is a continuing effort in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.

[0006] Polo-like kinases (Plk) belong to the serine/threonine kinase family, which are highly conserved between different species from yeast to humans (see Lowery DM et al, Oncogene 2005, 24; 248-. Plk kinases play multiple roles in the cell cycle, including entry into and control of progression through mitosis.

[0007] Plk1 was the most well identified of the Plk family members. Plk1 is widely expressed and is most abundant in tissues with high mitotic indices. The protein levels of Plk1 rise and peak in mitosis (Hamanaka, R et al, J Biol Chem 1995, 270, 21086-. The reported Plk1 substrates are all molecules known to regulate entry into and progression through mitosis, including CDC25C, cyclin B, p53, APC, BRCA2, and proteasomes. Plk1 is upregulated in a variety of cancer types, with expression levels correlated with disease severity (Macmillan, JC et al, Ann Surg Oncol 2001, 8, 729-. Plk1 is an oncogene capable of transforming NIH-3T3 cells (Smith, MR et al, Biochem Biophys Res Commun 1997, 234, 397-405). Plk1 reduces tumor Cell proliferation and viability in vitro due to either siRNA (antisense, microinjected antibody) deletion or inhibition or transfection of a dominant negative component of Plk1 into cells (Guan, R et al, Cancer Res 2005, 65, 2698-. Tumor cells deficient in Plk1 have spindle checkpoints activated as well as defects in spindle production, chromosome alignment and segregation, and cytokinesis. Loss of viability has been reported to be the result of induction of apoptosis. In contrast, normal cells were reported to maintain viability following the absence of Plk 1. In vivo Plk1 knockdown by siRNA or the use of a dominant negative component would cause growth inhibition or regression of tumors in xenograft models.

[0008] Plk2 is expressed primarily during the G1 phase of the cell cycle, and is localized to centrosomes in interphase cells. Plk2 knockout mice develop normally, are fertile, and have normal survival rates, but are about 20% lower than wild-type mice. Cells from the knockout animals progress more slowly through the Cell cycle than normal mice (Ma, S et al, Mol Cell Biol 2003, 23, 6936-6943). Plk2 blocked centromere replication due to siRNA deletion or transfection of kinase-inactive mutants into cells. Down-regulation of Plk2 also sensitizes tumor cells to paclitaxel, promoting mitotic catastrophe, in part by inhibition of the p53 response (Burns TF et al, Mol Cell Biol 2003, 23, 5556-.

[0009] Plk3 is expressed throughout the cell cycle, increasing from G1 to mitosis. Expression is upregulated in hyperproliferative ovarian and breast cancers and is associated with poor prognosis (Weichet, W et al, Br J Cancer 2004, 90, 815-. In addition to regulation of mitosis, Plk3 is believed to be involved in golgi fission and DNA-damage responses during the cell cycle. Inhibition of dominant negative expression of Plk3 was reported to promote p 53-independent apoptosis after DNA damage, inhibiting tumor cell colony formation (Li, Z et al, J Biol Chem 2005, 280, 16843-.

[0010] Plk4 is structurally more distinct from other Plk family members. The deletion of this kinase leads to apoptosis in cancer cells (Li, J et al, Neopalasia 2005, 7, 312-. Mice with the Plk4 knockout stopped at E7.5, most cells were mitotic, and chromosomes were partially sequestered (Hudson, JW et al, Current Biology 2001, 11, 441-.

[0011] Molecules of the protein kinase family have been implicated in the growth, proliferation and survival of tumor cells. Therefore, there is an urgent need to develop compounds useful as protein kinase inhibitors. There is substantial evidence that Plk kinase is essential for cell differentiation. Cell cycle arrest is a clinically proven means of inhibiting tumor cell proliferation and viability. Therefore, there is a need to develop compounds useful as inhibitors of Plk family protein kinases (e.g., Plk1, Plk2, Plk3, and Plk4) that will inhibit proliferation, reduce tumor cell viability, and in particular, there is a strong medical need to develop new cancer treatments.

Summary of The Invention

[0012] The compounds of the present invention and pharmaceutically acceptable compositions thereof are useful as inhibitors of protein kinases. In some embodiments, these compounds are useful as inhibitors of PLK protein kinases; in some embodiments, an inhibitor of PLK1 protein kinase. These compounds have formula I as defined herein or a pharmaceutically acceptable salt thereof.

[0013] These compounds and pharmaceutically acceptable compositions thereof are useful for treating or preventing a variety of diseases, disorders or conditions, including but not limited to autoimmune, inflammatory, proliferative or hyperproliferative diseases, neurodegenerative diseases, or immunologically-mediated diseases. The compounds provided by the invention are also useful in kinase studies in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such kinases; and comparative evaluation of novel kinase inhibitors.

Detailed description of the invention

The present invention describes compounds of formula I:

wherein

R¹Is H, C_1-6Aliphatic radicals or C_3-6A cycloaliphatic group;

g is-C (R)₂-or-O-;

l is C_0-3Aliphatic radical, optionally substituted by 0 to 3J^LSubstitution;

ring A is triazolyl, optionally substituted with 0-3J^ASubstitution;

ring B is 5-6 memberedAn aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S; ring B is optionally substituted with 0-5J^BSubstituted, and optionally fused, to ring B';

ring B' is a 5-8 membered aromatic or non-aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S; ring B' is optionally substituted with 0-4J^B′Substitution;

each J^A、J^BAnd J^B′Independently is C_1-6Haloalkyl, halo, NO₂CN, Q or-Z-Q;

z is independently C_1-6Aliphatic radicals, optionally substituted by 0 to 3 occurrences of-NR-, -O-, -S-, -C (O) -, -C (═ NR) -, -C (═ NOR) -, -SO-or-SO₂-an interrupt; each Z is optionally substituted by 0-2J^ZSubstitution;

q is H; c_1-6An aliphatic group; a 3-8 membered aromatic or non-aromatic monocyclic ring having 0-3 heteroatoms independently selected from O, N and S; or an 8-12 membered aromatic or non-aromatic bicyclic ring system having 0-5 heteroatoms independently selected from O, N and S; each Q is optionally substituted by 0-5J^QSubstitution;

each J^LAnd J^ZIndependently is H, halo, C_1-6Aliphatic radical, C_3-6Cycloaliphatic radical, NO₂、CN、-NH₂、-NH(C_1-4Aliphatic group), -N (C)_1-4Aliphatic radical)₂、-OH、-O(C_1-4Aliphatic group), -CO₂H、-CO₂(C_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic group) or halo (C)_1-4Aliphatic groups);

each J^QIndependently is M or-Y-M;

each Y is independently unsubstituted C_1-6Aliphatic radicals, optionally substituted by 0 to 3 occurrences of-NR-, -O-, -S-, -C (O) -, -SO-or-SO₂-an interrupt;

each M is independently H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic seriesRadical), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR′、SH、SR′、NH₂、NHR′、N(R′)₂、COH、COR′、CONH₂、CONHR′、CONR′₂、NHCOR′、NR′COR′、NHCONH₂、NHCONHR′、NHCON(R′)₂、SO₂NH₂、SO₂NHR′、SO₂N(R′)₂、NHSO₂R 'or NR' SO₂R′；

R is H or unsubstituted C_1-6An aliphatic group;

r' is unsubstituted C_1-6An aliphatic group; or two R' groups together with the atoms to which they are bonded form an unsubstituted 3-8 membered non-aromatic monocyclic ring having 0-1 heteroatoms independently selected from O, N and S.

[0014] Also included within the invention are pharmaceutically acceptable salts of all compounds of formula I, including but not limited to the compounds of table 1.

[0015]The compounds of the present invention include those generally described above, and the classes, subclasses, and species disclosed herein are further illustrated. As used herein, the following definitions should apply. For the purposes of the present invention, the chemical elements will be according to the CAS version of Handbook of chemistry and Physics, 75^thEd is used for identification. In addition, the general principles of Organic Chemistry are described in "Organic Chemistry", Thomas Sorrell, university science Books, Sausaltito: 1999 and "March's Advanced organic chemistry", 5^th Ed.，Ed.：Smith，M.B.and March，J.，John Wiley&Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0016] As described herein, the designated range of atomic numbers includes any integer therein. For example, a group having 1-4 atoms may have 1, 2, 3, or 4 atoms.

[0017] As described herein, the compounds of the invention may be optionally substituted with one or more substituents, such as those set forth generally above, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the term "optionally substituted" is used interchangeably with the term "substituted or unsubstituted. In general, the term "substituted," whether preceded by the term "optionally," means that a hydrogen radical in a given structure is replaced with a radical that is designated as a substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. Substituent combinations contemplated by the present invention are preferably those that form stable or chemically feasible compounds.

[0018] The term "stable" as used herein means compounds that are substantially unchanged when subjected to the conditions used for their preparation, detection, preferably recovery, purification, and for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that remains substantially unchanged in the absence of moisture or other chemically reactive conditions at a temperature of 40 ℃ or less for at least one week.

[0019] The term "aliphatic group" or "aliphatic group" as used herein means a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more units of unsaturation, which has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched substituted or unsubstituted alkyl, alkenyl, or alkynyl groups. Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl, ethynyl, and tert-butyl.

[0020]The term "cycloaliphatic" (alternatively "carbocycle" or "carbocyclyl" or "cycloalkyl", etc.) denotes a monocyclic C₃-C₈Hydrocarbons or bicyclic radicals C₈-C₁₂A hydrocarbon, which is fully saturated or contains one or more units of unsaturation, but is not aromatic, has a single point of attachment to the rest of the molecule, wherein any single ring in said bicyclic ring system has from 3 to 7 members. Suitable cycloaliphatic groups include, but are not limited to, cycloalkyl and cycloalkenyl. Specific examples include, but are not limited to, cyclohexyl, cyclopropenyl, and cyclobutyl.

[0021] The terms "heterocycle", "heterocyclyl" or "heterocyclic" and the like as used herein, denote non-aromatic monocyclic, bicyclic or tricyclic ring systems in which one or more ring members are independently selected heteroatoms. In some embodiments, a "heterocycle", "heterocyclyl", or "heterocyclic" group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

[0022] Suitable heterocycles include, but are not limited to, 3-1H-benzimidazol-2-one, 3- (1-alkyl) -benzimidazol-2-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothienyl, 3-tetrahydrothienyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropyrazinyl, 2-tetrahydropyrazinyl, 3-tetrahydropyrazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiophenyl, benzodithiane, and 1, 3-dihydro-imidazol-2-one.

[0023] Cyclic groups (e.g., cycloaliphatic and heterocyclic) can be linearly fused, bridged, or spiro.

[0024]The term "heteroatom" denotes one or more oxygen, sulfur, nitrogen or phosphorus (including any oxidized form of nitrogen, sulfur, phosphorus or silicon; quaternized forms of any basic nitrogen or heterocyclic ring substitutable nitrogen, e.g. N (as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺(as in N-substituted pyrrolidinyl)).

[0025] The term "unsaturated" as used herein means a moiety having one or more units of unsaturation.

[0026] The term "non-aromatic" as used herein describes a saturated or partially unsaturated ring.

[0027] The term "aromatic" as used herein describes a fully saturated ring.

[0028] The term "alkoxy" or "thioalkyl" as used herein means an alkyl group, as defined above, attached to the bulk carbon chain through an oxygen ("alkoxy") or sulfur ("thioalkyl") atom.

[0029] The terms "haloalkyl", "haloalkenyl" and "haloalkoxy" denote alkyl, alkenyl or alkoxy groups, as the case may be, substituted with one or more halogen atoms. The terms "halogen", "halo" and "hal" denote F, Cl, Br or I.

[0030] The term "aryl", used alone or as part of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl", denotes monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". The term "aryl" also denotes heteroaryl ring systems as defined below.

[0031] The term "heteroaryl", used alone or as part of a larger portion of "heteroaralkyl" or "heteroarylalkoxy", denotes monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring" or the term "heteroaromatic". Suitable heteroaryl rings include, but are not limited to, 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, benzofuranyl, benzothienyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 3-triazolyl, 1, 2, 3-thiadiazolyl, 1, 3, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, purinyl, pyrazinyl, 1, 3, 5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl).

[0032] The terms "protecting group" and "protecting group" as used herein refer to an ingredient used to temporarily block one or more desired reactive sites in a polyfunctional compound. In certain embodiments, the protecting group has one or more, or preferably all, of the following characteristics: a) selective reaction in good yield to give a protected substrate which is stable to reactions occurring at one or more other reactive sites; and b) selectively removed in good yield by a reagent that does not attack the regenerated functional group. Exemplary protecting groups are found in Greene, t.w., Wuts, p.g. in "Protective group in Organic Synthesis", Third Edition, John Wiley & Sons, New York: 1999 and other versions of this document, the entire contents of which are incorporated herein by reference. The term "nitrogen protecting group" as used herein means an ingredient used to temporarily block one or more desired nitrogen reactive sites in a polyfunctional compound. Preferred nitrogen protecting groups also have the above characteristics, and certain exemplary nitrogen protecting groups are also described in Chapter 7 in Greene, t.w., Wuts, p.g. in "protective groups in Organic Synthesis", Third Edition, John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

[0033]In some embodiments, the alkyl or aliphatic chain may optionally be interrupted by another atom or group. This means that the methylene units of the alkyl or aliphatic chain are optionally replaced by said other atoms or groups. Examples of such atoms or groups would include, but are not limited to, -NR-, -O-, -S-, -CO₂-、-OC(O)-、-C(O)CO-、-C(O)-、-C(O)NR-、-C(＝N-CN)、-NRCO-、-NRC(O)O-、-SO₂NR-、-NRSO₂-、-NRC(O)NR-、-OC(O)NR-、-NRSO₂NR-, -SO-or-SO₂-, wherein R is as defined herein. Alternative substitutions, unless otherwise specified, produce chemically stable compounds. Alternative substitutions may occur within the strand, as well as at the end of the strand; i.e. at the connection point and/or also at the end. The two alternative substitutions may also be adjacent to each other within the chain, as long as a chemically stable compound results. Optional interruptions or substitutions may also completely replace all carbon atoms in the chain. E.g. C₃Aliphatic groups may optionally be interrupted by or replaced by-NR-, -C (O) -and-NR-, yielding-NRC (O) NR- (urea).

[0034]Unless otherwise specified, if a substitution or interruption occurs at a terminus, a substitute atom is bonded to the terminal H. For example, if-CH₂CH₂CH₃Optionally interrupted by-O-, the resulting compound may be-OCH₂CH₃、-CH₂OCH₃or-CH₂CH₂OH。

[0035] Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, geometric, conformational or rotational mixtures of these compounds are within the scope of the invention.

[0036] Unless otherwise specified, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0037]Unless otherwise specified, a substituent may be free to rotate about any rotatable bond. For example, is depicted asThe substituents also represent

[0038]In addition, unless otherwise specified, the structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, except that hydrogen is replaced by deuterium or tritium or carbon is replaced by¹³C-or¹⁴C-enriched carbon instead of compounds having the structure of the present invention are within the scope of the present invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0039] The following abbreviations are used:

PG protecting group

LG leaving group

DCM dichloromethane

Ac acetyl group

DMF dimethyl formamide

EtOAc ethyl acetate

DMSO dimethyl sulfoxide

MeCN acetonitrile

TCA trichloroacetic acid

ATP adenosine triphosphate

EtOH ethanol

Ph phenyl

Me methyl group

Et Ethyl group

Bu butyl

DEAD azodicarboxylic acid diethyl ester

HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid

BSA bovine serum albumin

DTT dithiothreitol

MOPS 4-Morpholpropanesulfonic acid

NMR nuclear magnetic resonance

HPLC high performance liquid chromatography

LCMS liquid chromatography-mass spectrometry

TLC thin layer chromatography

Rt Retention time

[0040]In some embodiments of the invention, G is-C (R)₂-. In other embodiments, G is O.

[0041]In some embodiments, R¹Is H.

[0042] In some embodiments, ring A is selected from

[0043]In some embodiments, ring B is a 5-6 membered aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S. In some embodiments, ring B is a 5-membered ring. In other embodiments, ring B is a 6-membered ring. In some embodiments, ring B is fused to ring B'. In some embodiments, ring B' is a 5-6 membered aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S. In all of these embodiments, ring B is optionally substituted with 0-5J^BSubstituted, ring B' is optionally substituted with 0-4J^B′And (4) substitution.

[0044]In some embodiments, J^AIs H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂And R is shown in the specification. In some embodiments, J^AIs H.

[0045]In some embodiments, J^BIs H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂R。

[0046]In other embodiments, J^B′Is H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂R。

In some embodiments, L is C_0-3Alkyl, optionally substituted with 0-3J^LAnd (4) substitution. In other embodiments, L is C_1-3Alkyl, optionally substituted with 0-3J^LAnd (4) substitution. In other embodiments, L is-CH (CH)₃)-。

[0047]In some embodiments, ring B is phenyl, optionally substituted with 0-5J^BAnd (4) substitution.

[0048] In some embodiments, the variables are as depicted for the compounds in table 1.

[0049] In some embodiments, the compounds of the present invention are shown in table 1:

TABLE 1

General synthetic method

[0050] The compounds of the invention can generally be prepared by those methods depicted in the following general schemes and the preparations that follow. All variables in the following schemes are as defined herein, unless otherwise indicated.

Scheme 1

[0051]Scheme 1 above shows a general synthetic route for the preparation of compounds of the present invention, wherein ring a is attached to the thiophene core via a nitrogen atom. Conjugation of Michael acceptor 1 (prepared by a method similar to that reported in Synthesis, (10), 847-850, 1984) to additionReaction, wherein ring A is a nitrogen-containing ring, produces 2. The 3-hydroxy group of 2 is then derivatized with the appropriate functional group under Mitsunobu conditions to provide 3. Finally, the ester moiety in 3 is converted to an amide under suitable amide forming conditions to provide the compounds of the invention.

Scheme 2

[0052] Scheme 2 above shows a general synthetic route to the preparation of certain ring a derivatives. Cyanamide 5 was treated with aldehyde and hydrazine (in a manner similar to that reported in Synthesis, (3), 222-. It may then undergo further functionalization as described in scheme 1 to give the compounds of the invention.

[0053] Accordingly, the present invention also provides methods for preparing the compounds of the present invention.

[0054] One embodiment of the present invention provides a process for preparing a compound of formula I:

wherein G is O, R¹Is H, ring A, ring B, J^A、J^BAnd L is as defined herein, comprising reacting a compound of formula 3

Wherein G is O, R¹Is C_1-6Aliphatic radical, ring A, ring B, J^A、J^BAnd L is as defined herein, and,

under suitable amide forming conditions to produce the compound of formula I. Suitable amide forming conditions are known to those skilled in the art and can be found in "March's Advanced organic chemistry". Examples of suitable amide forming conditions include, but are not limited to, NH₃Carboxylic acid methyl ester was heated in the presence of MeOH.

[0055] One embodiment of the present invention provides a process for preparing a compound of formula 3:

wherein G is O, ring A, ring B, J^A、J^BAnd L is as defined herein, comprising reacting a compound of formula 2:

andcarrying out reaction; wherein LG is a suitable leaving group, L, Ring B and J^BIs as defined herein; under suitable O-C bond coupling conditions to produce the compound of formula 3.

[0056] Another embodiment provides a method of preparing a compound of formula 2:

comprising reacting a compound of formula 1:

addition ofWherein ring A is an aromatic ring containing a nitrogen atom capable of nucleophilic attack, J^AIs as defined herein;

the compound of formula 2 is produced via suitable conjugate addition conditions. Examples of aromatic rings containing a nitrogen atom capable of nucleophilic attack are

Suitable conjugate addition conditions include, but are not limited to, combining 2 equivalents at room temperatureWith a compound of formula 8 in DCM; combining 1.15 equivalents at 110 ℃With the compound of formula 8 in acetonitrile/DMF overnight.

[0057] Another embodiment provides a method of preparing a compound of formula 6:

comprising reacting a compound of formula 5:

treatment with aldehyde and hydrazine (in a manner similar to that reported in Synthesis, (3), 222-.

[0058]In some embodiments of the present invention, the substrate is,is a compound of formula 6.

[0059] The present invention provides compounds that are useful in the treatment of diseases, disorders, and conditions, including but not limited to, autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immunologically-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone-related diseases, allergies, asthma, and alzheimer's disease. Another aspect of the invention provides protein kinase inhibitor compounds, and thus are useful for treating diseases, disorders, and conditions, as well as other uses described herein. In another aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the compounds described herein, optionally together with a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents.

[0060] It will also be appreciated that certain compounds of the invention can be present in free form for use in therapy, or as appropriate, a pharmaceutically acceptable salt or pharmaceutically acceptable derivative thereof.

[0061] As used herein, a "pharmaceutically acceptable derivative" is an adduct or derivative that upon administration to a patient in need thereof is capable of providing, directly or indirectly, a compound of the present invention or an inhibitory active metabolite or residue thereof. Examples of pharmaceutically acceptable derivatives include, but are not limited to, esters and salts of such esters.

[0062] The term "pharmaceutically acceptable salt" as used herein, means salts of the compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio.

[0063] Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts are described in detail, for example, in j.pharmaceutical Sciences, 1977, 66, 1-19, by s.m.berge et al, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. These salts may be prepared in situ during the final isolation and purification of the compounds. Acid addition salts can be prepared as follows: 1) reacting the free base form of the purified compound with a suitable organic or inorganic acid, and 2) isolating the salt formed.

[0064]Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, embonate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄And (3) salt. The invention also encompasses quaternization of any basic nitrogen-containing group of the compounds as disclosed herein. By means of such quaternization, products which are soluble or dispersible in water or oil can be obtained.

[0065] Base addition salts can be prepared as follows: 1) reacting the acid form of the purified compound with a suitable organic or inorganic base, and 2) isolating the salt formed. Base addition salts include alkali metal or alkaline earth metal salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and amine cations, generated using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Other acids and bases, although not pharmaceutically acceptable per se, may be used to prepare salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid or base addition salts.

[0066] As described herein, the pharmaceutically acceptable compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle, which, as used herein, includes any and all solvents, diluents, or other liquid excipients, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980) disclose various carriers for formulating pharmaceutically acceptable compositions and known techniques for their preparation. The use of any conventional carrier medium is contemplated within the scope of the present invention, except as being incompatible with the compounds of the present invention, e.g., producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition.

[0067] Some examples of materials capable of serving as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins, such as serum albumin; buffer substances, such as phosphates; glycine; sorbic acid or potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon dioxide; magnesium trisilicate; polyvinylpyrrolidone; a polyacrylate; waxes; polyethylene-polypropylene oxide-block polymers; lanolin; sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; crushed tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and anti-oxidizing agents may also be present in the composition, according to the judgment of the person skilled in the art.

[0068] One aspect of the invention provides a method of treating or lessening the severity of a disease selected from the group consisting of: an autoimmune disease, an inflammatory disease, a proliferative or hyperproliferative disease (e.g., cancer), an immunologically-mediated disease, a bone disease, a metabolic disease, a neurological or neurodegenerative disease, a cardiovascular disease, an allergy, asthma, alzheimer's disease, or a hormone-related disease, comprising administering to a subject in need thereof an effective amount of the compound or a pharmaceutically acceptable composition comprising the compound. The term "cancer" includes, but is not limited to, the following cancers: mammary gland; an ovary; the cervix; the prostate; testis, urogenital tract; an esophagus; larynx, glioblastoma; neuroblastoma; the stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma; a bone; colon, adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma; melanoma; a sarcoma; bladder cancer; liver cancer and biliary tract; kidney cancer; myeloid disorders; lymphoid disorders, hodgkin's disease, hair cells; oral cavity and pharynx (oral), lip, tongue, mouth, pharynx; the small intestine; colon-rectum, large intestine, rectum; the brain and central nervous system; and leukemia.

[0069] In certain embodiments, an "effective amount" of a compound or pharmaceutically acceptable composition is an amount effective to treat the disease. The compounds and compositions according to the methods of the present invention may be administered in any amount and by any route of administration that is effective in treating or lessening the severity of the disease. In some embodiments, the disease is selected from the group consisting of a proliferative disorder, a neurodegenerative disorder, an autoimmune disorder, an inflammatory disorder, and an immunologically-mediated disorder. In some embodiments, the disease is a proliferative disorder. In some embodiments cancer.

[0070] In other embodiments of the invention, the disease is a protein kinase mediated disorder. In some embodiments, the protein kinase is PLK.

[0071] The term "protein kinase-mediated condition" as used herein refers to any disease or other deleterious condition in which a protein kinase is known to play a role. Such conditions include, without limitation, autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immunologically-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone-related diseases, allergies, asthma, and alzheimer's disease.

[0072] The term "PLK-mediated disorder" as used herein means any disease or other deleterious disorder in which PLK plays a role. Such conditions include, without limitation, proliferative disorders (e.g., cancer), neurodegenerative disorders, autoimmune disorders, inflammatory disorders, and immunologically-mediated disorders.

[0073] In some embodiments, the compounds and compositions of the present invention are inhibitors of protein kinases. The compounds and compositions of the invention are particularly useful for treating or lessening the severity of a disease, condition or disorder in which a protein kinase is implicated as an inhibitor of the protein kinase. In one aspect, the invention provides methods of treating or lessening the severity of a disease, disorder or condition in which a protein kinase is implicated. In another aspect, the invention provides a method of treating or lessening the severity of a disease, disorder or condition, wherein inhibition of enzyme activity is implicated in the treatment of the disease. In another aspect, the invention provides methods of treating or lessening the severity of a disease, disorder or condition with a compound that inhibits enzymatic activity by binding to a protein kinase. In some embodiments, the protein kinase is PLK.

[0074] The activity of the compounds used as protein kinase inhibitors in the present invention may be determined in vitro, in vivo or in a cell line. In vitro assays include measuring inhibition of kinase activity or ATPase activity of activated kinases. A selective in vitro assay quantifies the ability of an inhibitor to bind to a protein kinase. Binding of the inhibitor can be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/kinase complex and determining the amount of radiolabel bound, or by performing a competition experiment in which a new inhibitor is incubated with a kinase bound to a known radioligand.

[0075] The protein kinase inhibitor or a pharmaceutical salt thereof may be formulated into a pharmaceutical composition for administration to an animal or human. These pharmaceutical compositions, which comprise a protein kinase inhibitor in an amount effective to treat or prevent a kinase-mediated disorder and a pharmaceutically acceptable carrier, are another embodiment of the present invention. In some embodiments, the protein kinase-mediated disorder is a PLK-mediated disorder. In some embodiments, Plk 1-mediated disorders.

[0076] The exact amount of the compound required for treatment will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the infection, the particular drug, the manner of its administration, and the like. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein denotes physically discrete pharmaceutical units, as appropriate for the patient to be treated. It will be understood, however, that the total daily amount of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the particular compound employed; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed; and other factors well known in the medical arts. The term "patient" as used herein means an animal, preferably a mammal, most preferably a human.

[0077] The pharmaceutically acceptable compositions of the present invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, or drops), buccally, as an oral or nasal spray, and the like to humans and other animals, depending on the severity of the infection being treated. In certain embodiments, the compounds of the present invention may be administered orally or parenterally at a dosage level of from about 0.01mg/kg to about 50mg/kg, preferably from about 1mg/kg to about 25mg/kg, of the subject's body weight per day, one or more times a day, to achieve the desired therapeutic effect.

[0078] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0079] Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable carriers and solvents that may be employed are water, ringer's solution, U.S. p. and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids, such as oleic acid, may be used in the preparation of injectables.

[0080] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0081] In order to prolong the effect of the compounds of the invention, it is often desirable to delay absorption of the compounds following subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of a compound depends on its rate of dissolution, which in turn may depend on crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered compound form is achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are prepared by forming a microencapsulated matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues.

[0082] Rectal or vaginal compositions are preferably suppositories which can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active compound.

[0083] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, for example sodium citrate or dicalcium phosphate, and/or a) fillers or extenders, for example starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) wetting agents, for example glycerol, d) disintegrants, for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retarders, for example paraffin, f) absorption accelerators, for example quaternary ammonium compounds, g) wetting agents, for example cetyl alcohol and glycerol monostearate, h) absorbents, for example kaolin and bentonite, and i) lubricants, for example talc, calcium stearate, sodium silicate, and the like, Magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0084] Solid compositions of a similar type may also be employed as fillers in soft or hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as polymeric polyethylene glycols and the like. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be provided with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as polymeric polyethylene glycols and the like.

[0085] The active compound may also be in microencapsulated form, containing one or more of the above-mentioned excipients. Solid dosage forms such as tablets, dragees, capsules, pills and granules can be provided with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, for example sucrose, lactose or starch. Such dosage forms may also contain, under normal circumstances, other substances in addition to inert diluents, such as tableting lubricants and other tableting aids, for example magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0086] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers, as appropriate. Ophthalmic formulations, ear drops and eye drops are also encompassed within the scope of the present invention. In addition, the present invention encompasses the use of transdermal patches, which have the added advantage of controlling the delivery of compounds to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers may also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0087] In addition to the compounds of the present invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of the present invention may also be employed in compositions for the treatment or prevention of the above-mentioned conditions.

[0088] By "pharmaceutically acceptable derivative or prodrug" is meant any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of the invention that, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the invention or an inhibitory active metabolite or residue thereof. Particularly desirable derivatives and prodrugs are those which increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing orally administered compounds to be more readily absorbed into the blood), or which enhance delivery of the parent compound to a biological cavity (e.g., the brain or lymphatic system) relative to the parent species.

[0089] Pharmaceutically acceptable prodrugs of the compounds of the present invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts, and sulfonate esters.

[0090] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene-block polymers, polyethylene glycol, and wool fat.

[0091] Administration of the compositions of the invention may be oral, parenteral, inhalation spray, topical, rectal, nasal, buccal, vaginal or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally, or intravenously.

[0092] The sterile injectable dosage form of the compositions of the present invention may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. 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-butanediol. Among the acceptable carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any brand of fixed oil may be used, including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as tweens, spans and other emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0093] The pharmaceutically acceptable compositions of the present invention may be administered orally, and any orally acceptable dosage form includes, but is not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule dosage forms, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is mixed with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0094] Alternatively, the pharmaceutical compositions of the present invention may be administered in the form of suppositories for rectal administration. They may be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0095] The pharmaceutical compositions of the present invention may also be administered topically, particularly when the target of treatment includes sites or organs readily accessible by topical application, including diseases of the eye, skin or lower intestinal tract. Suitable topical formulations are readily prepared for each of these sites or organs.

[0096] Topical lower intestinal administration may be carried out using rectal suppositories (see above) or suitable enemas. Topical transdermal patches may also be used.

[0097] For topical administration, the pharmaceutical compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Topical carriers for the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0098] For ophthalmic purposes, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic pH adjusted sterile saline or, preferably, as solutions in isotonic pH adjusted sterile saline, both with or without preservatives, such as benzalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical composition may be formulated in an ointment, such as petrolatum.

[0099] The pharmaceutical compositions of the present invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents.

[00100] The amount of kinase inhibitor that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. Preferably, the compositions should be formulated so that a dose of between 0.01 and 100mg/kg body weight/day of inhibitor can be administered to a patient receiving these compositions.

[00101] It will also be understood that the specific dose and regimen of treatment for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the attending physician, and the severity of the particular disease being treated. The amount of inhibitor will also depend on the particular compound in the composition.

[00102] According to another embodiment, the present invention provides a method of treating or preventing a protein kinase-mediated disorder (in some embodiments, a PLK-mediated disorder), comprising the step of administering to a patient one of the above-described pharmaceutical compositions. The term "patient" as used herein means an animal, preferably a human.

[00103] Preferably, the method is for treating or preventing a condition selected from the group consisting of: cancers, such as breast, colon, prostate, skin, pancreas, brain, genitourinary tract, lymphatic system, stomach, larynx and lung cancers, including lung adenocarcinoma and small cell lung cancer; stroke, diabetes, melanoma, hepatomegaly, cardiac hypertrophy, alzheimer's disease, cystic fibrosis and viral disease or any of the specific diseases or conditions mentioned above.

[00104] Another aspect of the invention relates to inhibiting protein kinase activity in a patient, comprising administering to the patient a compound of formula I or a composition comprising the compound.

[00105] Depending on the particular protein kinase-mediated condition to be treated or prevented, additional agents that are normally administered to treat or prevent the condition may be administered with the inhibitors of the invention. For example, chemotherapeutic agents or other anti-proliferative agents may be combined with the protein kinase inhibitors of the present invention to treat proliferative diseases.

[00106] These additional drugs may be administered separately from the compound or composition containing the protein kinase inhibitor as part of a multiple dose regimen. Alternatively, these agents may be part of a single dosage form, mixed together with the protein kinase inhibitor in a single composition.

[00107] In some embodiments, the protein kinase inhibitor is a PLK kinase inhibitor. In other embodiments, the protein kinase inhibitor is a Plk1 kinase inhibitor.

[00108] The invention may also be used in those methods that do not involve administration to a patient.

[00109]One aspect of the invention relates to inhibiting protein kinase activity in a biological sample or patient, the method comprising contacting said biological sample with a compound of formula I or a composition comprising said compound. The term "biological sample" as used herein meansIn vitroOr fromIn vivoIncluding, without limitation, cell cultures and extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tearsLiquid or other body fluid or extract thereof.

[00110] Inhibition of protein kinase activity in a biological sample can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, and biological sample storage.

[00111] Another aspect of the invention relates to protein kinase studies in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such protein kinases; and comparative evaluation of novel protein kinase inhibitors. Examples of such uses include, but are not limited to, biological assays, such as enzymatic assays and cell-based assays.

[00112] The compounds of the invention can generally be prepared by methods known to those skilled in the art. These compounds can be analyzed by known methods including, but not limited to, LCMS (liquid chromatography mass spectrometry) and NMR (nuclear magnetic resonance). The compounds of the invention can also be tested according to these examples. It should be understood that the specific conditions shown below are merely examples and are not meant to limit the scope of conditions that can be used to prepare, analyze, or test the compounds of the present invention. Rather, the present invention also encompasses conditions known to those skilled in the art for preparing, analyzing and testing the compounds of the present invention.

Examples

[00113] The term "rt (min)" as used herein denotes the HPLC retention time in minutes, in relation to the compound. Unless otherwise indicated, the HPLC method used to obtain the reported retention times was as follows:

column: ACE C8 column, 4.6x150mm

Gradient: 0-100% acetonitrile + methanol 60: 40(20mM Tris phosphate)

Flow rate: 1.5 mL/min

And (3) detection: 225 nm.

[00114] Mass spectral samples were analyzed on a MicroMass Quattro Micro mass spectrometer operating in single MS mode and electrospray ionization mode. The sample was introduced to the mass spectrometer using chromatographic methods.

[00115]Recording at 400MHz using a Bruker DPX 400 instrument¹H-NMR spectrum. The following compounds of formula I were prepared and analyzed as follows.

Example 1：

3- [1- (2-chloro-phenyl) -ethoxy ] -5- (3-phenylamino- [1, 2, 4] triazol-1-yl) -thiophene-2-carboxylic acid amide (I-1)

The method A comprises the following steps: 3-hydroxy-5- (3-phenylamino- [1, 2, 4] triazol-1-yl) -thiophene-2-carboxylic acid methyl ester

[00116]Phenyl- (1H- [1, 2, 4)]Triazol-3-yl) -amine (646mg, 4.06mmol, 1.15Eq.) and methyl 2-chloro-3-oxo-2, 3-dihydro-thiophene-2-carboxylate (680mg, 3.53mmol, 1.0Eq.) were dissolved in anhydrous MeCN (10mL) and DMF (3mL) and heated at reflux for 18 h. After cooling to ambient temperature, the resulting precipitate was isolated by filtration and washed with DCM to give the sub-title compound as a brown solid (415mg, 1.31mmol 37%);¹HNMR(400MHz，CDCl₃)δ3.77(3H，s)，6.89(1H，t)，7.11(1H，s)，7.31(2H，t)，7.57(2H，t)，9.15(1H，s)，9.70(1H，s)，10.78(1H，s)。

the method B comprises the following steps: 3- [1- (2-chloro-phenyl) -ethoxy ] -5- (3-phenylamino- [1, 2, 4] triazol-1-yl) -thiophene-2-carboxylic acid methyl ester

[00117]Triphenylphosphine (124mg, 0.47mmol, 1.5Eq.) was added to a stirred solution of 1- (2-chloro-phenyl) -ethanol (74mg, 0.47mmol, 1.5Eq.) in anhydrous THF (5mL) at 0 ℃ under nitrogen. The reaction was stirred at this temperature for 30 minutes, then diethyl azodicarboxylate (75 μ L, 0.47mmol, 1.5Eq.) was added dropwise and the reaction stirred at 0 ℃ for an additional 1 hour. Adding 3-hydroxy-5- (3-phenylamino- [1, 2, 4)]A solution of triazol-1-yl) -thiophene-2-carboxylic acid methyl ester (100mg, 0.32mmol, 1.0Eq.) in anhydrous DMF (2mL) was stirred at 0 ℃ for an additional 1 hour, then warmed to ambient temperature overnight. The crude reaction mixture was partitioned between EtOAc (50mL) and water (50 mL). The organic phase was washed with water (2 × 20mL) and dried (MgSO)₄) And concentrated in vacuo. The crude product was purified by column chromatography (ISCO company)^TM12g column, 0-100% EtOAc/petroleum ether) to give the sub-title compound as an off-white solid (88mg, 0.19mmol, 73%);¹H NMR(400MHz，CDCl₃)δ1.74(3H，d)，3.93(3H，s)，5.84(1H，q)，6.70(1h，br s)，6.74(1H，s)，7.02(1H，t)，7.21-7.40(5H，m)，7.51(1H，d)，7.63(1H，d)，8.12(1H，s)。

the method C comprises the following steps: 3- [1- (2-chloro-phenyl) -ethoxy ] -5- (3-phenylamino- [1, 2, 4] triazol-1-yl) -thiophene-2-carboxylic acid amide (I-1)

[00118]Reacting 3- [1- (2-chloro-phenyl) -ethoxy]-5- (3-phenylamino- [1, 2, 4)]Triazol-1-yl) -thiophene-2-carboxylic acid methyl ester (66mg, 0.15mmol, 1.0Eq.) was suspended in 7M NH₃In MeOH (10mL), heated to 100 ℃ in a pressure tube for 3 days. The solvent was removed in vacuo and the product isolated by column chromatography (10% MeOH/DCM) followed by trituration from ether to give the title compound as an off-white solid (17mg, 0.04mmol, 27%);¹H NMR(400MHz，d-6DMSO)δ1.72(3H，d)，5.88(1H，q)，6.88(1H，t)，7.03(1H，br s)，7.26(1H，s)，7.30(2H，t)，7.36-7.43(2H，m)，7.52(1H，dd)，7.57(2H，d)，7.66(1H，dd)，7.75(1H，br s)，9.02(1H，br s)，9.65(1H，s)。

example 2:

3- [1- (2-chloro-phenyl) -ethoxy ] -5- [3- (2, 2-difluoro-benzo [1, 3] dioxol-4-ylamino) - [1, 2, 4] triazol-1-yl ] -thiophene-2-carboxylic acid amide (I-2)

The method D comprises the following steps: (2, 2-difluoro-benzo [1, 3] dioxol-4-yl) - (1H- [1, 2, 4] triazol-3-yl) -amine

[00119]Reacting 2, 2-difluoro-benzo [1, 3]]Dioxol-4-yl-cyanamide (cyanamide) (1.0g, 5.05mmol, 1.0Eq.) was dissolved in MeOH (50mL) and water (10 mL). Hydrazine hydrate (245 μ L, 5.05mmol, 1.0Eq.) followed by 32 wt% aqueous formaldehyde (474mL, 5.05mmol, 1.0Eq.) was added and the reaction was stirred at ambient temperature overnight. The solvent was removed in vacuo and the residue was recrystallized from MeOH to give the sub-title compound as an off-white solid (516mg, 2.15mmol, 42%);¹H NMR(400MHz，d-6DMSO)δ6.88(1H，d)，7.10(1H，t)，7.72(1H，d)，8.09(1H，br s)，9.25(1H，br s)，13.20(1H，br s)；¹⁹f NMR (376MHz, d-6DMSO, decoupled protons) delta-49.1.

[00120]This intermediate was used in the sequence described for methods A-C to give the title compound as an off-white solid (19mg, 0.04mmol, 19%);¹H NMR(400MHz，d-6DMSO)δ1.72(3H，d)，5.87(1H，q)，7.00(2H，d+br s)，7.18(1H，t)，7.29(1H，s)，7.32-7.42(2H，m)，7.51(1H，dd)，7.64-7.68(2H，m)，7.93(1H，br s)，9.04(1H，s)，9.75(1H，s)；¹⁹f NMR (376MHz, d-6DMSO, decoupled protons) delta-40.1.

Example 3:

5- [3- (3-chloro-phenylamino) - [1, 2, 4] triazol-1-yl ] -3- [1- (2-chloro-phenyl) -ethoxy ] -thiophene-2-carboxylic acid amide (I-3)

[00121]Prepared from 3-chloro-phenyl-cyanamide using methods a-D described above, purified by trituration from EtOAc to give the title compound as a pale yellow solid (5mg, 0.01mmol, 5%);¹H NMR(400MHz，d-6DMSO)δ1.72(3H，s)，5.89(1H，q)，6.92(1H，dd)，7.04(1H，br s)，7.27(1H，s)，7.29-7.53(5H，m)，7.65-7.70(2H，m)，7.70(1h，br s)，9.05(1H，s)，9.90(1H，s)。

#	MS+	1HNMR	HPLCRt/min
				I-1	440.35	1HNMR(DMSO)1.72(3H，d)，5.88(1H，q)，6.88(1H，t)，7.03(1H，br s)，7.25-7.31(3H，m)，7.36-7.43(2H，m)，7.52(1H，d)，7.57(2H，d)，7.66(1H，dd)，7.75(1H，br s)，9.02(1H，br s)，9.65(1H，s)；	9.65
I-2	520.70	1HNMR(DMSO)1.72(3H，d)，5.87(1H，q)，7.00(2H，d+br s)，7.18(1H，t)，7.29(1H，s)，7.32-7.42(2H，m)，7.51(1H，dd)，7.64-7.68(2H，m)，7.93(1H，brs)，9.04(1H，s)，9.75(1H，s)	10.4
				I-3	474.70	1HNMR(DMSO)1.72(3H，s)，5.89(1H，q)，6.92(1H，dd)，7.04(1H，br s)，7.27(1H，s)，7.29-7.53(5H，m)，7.65-7.70(2H，m)，7.70(1h，br s)，9.05(1H，s)，9.90(1H，s)	10.3

example 4: PLK assay

[00122] The compounds of the invention can be evaluated as human PLK kinase inhibitors using the following assay.

Plk1 inhibition assay：

[00123]Compounds were screened for their ability to inhibit Plk1 using a radioactive phosphate binding assay. At 25mM HEPES (pH 7.5), 10mM MgCl₂The assay was performed in a mixture with 1mM DTT. The final substrate concentration was 50. mu.M [ gamma. -33P ]]ATP(136mCi 33P ATP/mmol ATP，Amersham Pharmacia Biotech/Sigma Chemicals)And 10. mu.M peptide (. DELTA.332-443 of SAM68 protein). The assay was performed in the presence of 15nM Plk1(A20-K338) at 25 ℃. Assay stock buffer solutions were prepared containing all reagents as listed above, with the exception of ATP and the test compound of interest. 30 μ L of stock solution was placed in 96-well plates followed by the addition of 2 μ L of DMSO stock solution, containing serial dilutions of test compound (usually starting at a final concentration of 10 μ M, in 2-fold serial dilutions), in duplicate (final DMSO concentration 5%). The plates were preincubated at 25 ℃ for 10 min, and 8. mu.L of [ gamma. -33P ] was added]ATP (final concentration 50. mu.M) initiates the reaction.

[00124] After 60 minutes, 100. mu.L of 0.14M phosphoric acid was added to terminate the reaction. The multi-sieve phosphocellulose filter 96-well plates (Millipore, Cat No. MAPN0B50) were pretreated with 100. mu.L of 0.2M phosphoric acid, and then 125. mu.L of the terminated assay mixture was added. The plate was washed with 4X 200. mu.L 0.2M phosphoric acid. After drying, 100 μ LOptiphase 'SuperMix' Liquid Scintillation cocktail reagent (Perkin Elmer) was added to the wells, followed by Scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

[00125] After removing the average background value for all data points, ki (app) data was calculated from nonlinear regression analysis of the initial rate data using the Prism Software package (GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

Plk1 inhibition assay：

[00126]Compounds were screened for their ability to inhibit Plk1 using a radioactive phosphate binding assay. At 25mM HEPES (pH 7.5), 10mM Mg l₂0.1% BSA in a mixture with 2mM DTT. The final substrate concentration was 150. mu.M [ gamma-33P ]]ATP (115mCi33P ATP/mmol ATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 300. mu.M peptide (KKKISDELMDATFADQEAK). The assay was performed in the presence of 4nM Plk1 at 25 ℃. Assay stock buffer solutions were prepared containing all reagents as listed above, with the exception of ATP and the test compound of interest. Place 30. mu.L of stock solution in 96-well plates, followed by addition of 2. mu.L of DMSO stock solutionSerial dilutions (usually starting at a final concentration of 10 μ M, diluted in 2-fold series) of the test compound were included in duplicate (final DMSO concentration 5%). The plates were preincubated at 25 ℃ for 10 min, and 8. mu.L of [ gamma. -33P ] was added]ATP (final concentration 150. mu.M) initiated the reaction.

[00127] After 90 minutes, 100. mu.L of 0.14M phosphoric acid was added to terminate the reaction. The multi-sieve phosphocellulose filter 96-well plates (Millipore, Cat No. MAPN0B50) were pretreated with 100. mu.L of 0.2M phosphoric acid, and then 125. mu.L of the terminated assay mixture was added. The plate was washed with 4X 200. mu.L 0.2M phosphoric acid. After drying, 100 μ L Optiphase 'Supermix' Liquid Scintillation cocktail reagent (Perkin Elmer) was added to the wells, followed by Scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

[00128] After removing the average background value for all data points, ki (app) data was calculated from nonlinear regression analysis of the initial rate data using the Prism Software package (GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

[00129] In general, the compounds of the invention are effective in terms of inhibition of Plk 1. The following compounds showed Ki between 10nM and 100nM in the radioactive binding assay: i-1, I-2 and I-3.

Plk2 inhibition assay：

[00130]Compounds were screened for their ability to inhibit Plk2 using a radioactive phosphate binding assay. At 25mM HEPES (pH 7.5), 10mM MgCl₂0.1% BSA in a mixture with 2mM DTT. The final substrate concentration was 200. mu.M [ gamma. -33P ]]ATP (57mCi33P ATP/mmol ATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 300. mu.M peptide (KKKISDELMDATFADQEAK). The assay was performed in the presence of 25nM Plk2 at 25 ℃. Assay stock buffer solutions were prepared containing all reagents as listed above, with the exception of ATP and the test compound of interest. Place 30. mu.L of the stock solution in a 96-well plate, followed by the addition of 2. mu.L of DMSO stock solution, which contains serial dilutions of the test compound (usuallyStarting at a final concentration of 10 μ M, diluted in a 2-fold series), in duplicate (final DMSO concentration 5%). The plates were preincubated at 25 ℃ for 10 min, and 8. mu.L of [ gamma. -33P ] was added]ATP (final concentration 200. mu.M) initiated the reaction.

[00131] After 90 minutes, 100. mu.L of 0.14M phosphoric acid was added to terminate the reaction. The multi-sieve phosphocellulose filter 96-well plates (Millipore, Cat No. MAPN0B50) were pretreated with 100. mu.L of 0.2M phosphoric acid, and then 125. mu.L of the terminated assay mixture was added. The plate was washed with 4X 200. mu.L 0.2M phosphoric acid. After drying, 100 μ L Optiphase 'Supermix' Liquid Scintillation cocktail reagent (Perkin Elmer) was added to the wells, followed by Scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

[00132] After removing the average background value for all data points, ki (app) data was calculated from nonlinear regression analysis of the initial rate data using the Prism Software package (GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

Plk3 inhibition assay：

[00133]Compounds were screened for their ability to inhibit Plk3 using a radioactive phosphate binding assay. At 25mM HEPES (pH 7.5), 10mM MgCl₂The assay was performed in a mixture with 1mM DTT. The final substrate concentration was 75. mu.M [ gamma. -33P ]]ATP (60mCi 33P ATP/mmolATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 10. mu.M peptide (SAM68 protein. DELTA. 332-443). The assay was performed in the presence of 5nM Plk3(S38-A340) at 25 ℃. Assay stock buffer solutions were prepared containing all reagents as listed above, with the exception of ATP and the test compound of interest. 30 μ L of stock solution was placed in 96-well plates followed by the addition of 2 μ L of DMSO stock solution, containing serial dilutions of test compound (usually starting at a final concentration of 10 μ M, in 2-fold serial dilutions), in duplicate (final DMSO concentration 5%). The plates were preincubated at 25 ℃ for 10 min, and 8. mu.L of [ gamma. -33P ] was added]ATP (final concentration 75. mu.M) initiated the reaction.

[00134] After 60 minutes, 100. mu.L of 0.14M phosphoric acid was added to terminate the reaction. The multi-sieve phosphocellulose filter 96-well plates (Millipore, Cat No. MAPN0B50) were pretreated with 100. mu.L of 0.2M phosphoric acid, and then 125. mu.L of the terminated assay mixture was added. The plate was washed with 4X 200. mu.L 0.2M phosphoric acid. After drying, 100 μ L Optiphase 'Supermix' Liquid Scintillation cocktail reagent (Perkin Elmer) was added to the wells, followed by Scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

[00135] After removing the average background value for all data points, ki (app) data was calculated from nonlinear regression analysis of the initial rate data using the Prism Software package (GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

Plk4 inhibition assay：

[00136]Compounds were screened for their ability to inhibit Plk4 using a radioactive phosphate binding assay. At 8mM MOPS (pH 7.5), 10mM MgCl₂0.1% BSA in a mixture with 2mM DTT. The final substrate concentration was 15. mu.M [ gamma. -33P ]]ATP (227mCi 33PATP/mmol ATP, Amersham Pharmacia Biotech/Sigma Chemicals) and 300. mu.M peptide (KKKMDATFADQ). The assay was performed in the presence of 25nM Plk4 at 25 ℃. Assay stock buffer solutions were prepared containing all reagents as listed above, with the exception of ATP and the test compound of interest. 30 μ L of stock solution was placed in 96-well plates followed by the addition of 2 μ L of DMSO stock solution, containing serial dilutions of test compound (usually starting at a final concentration of 10 μ M, in 2-fold serial dilutions), in duplicate (final DMSO concentration 5%). The plates were preincubated at 25 ℃ for 10 min, and 8. mu.L of [ gamma. -33P ] was added]ATP (final concentration 15. mu.M) initiated the reaction.

[00137] After 180 minutes, 100. mu.L of 0.14M phosphoric acid was added to terminate the reaction. The multi-sieve phosphocellulose filter 96-well plates (Millipore, Cat No. MAPN0B50) were pretreated with 100. mu.L of 0.2M phosphoric acid, and then 125. mu.L of the terminated assay mixture was added. The plate was washed with 4X 200. mu.L 0.2M phosphoric acid. After drying, 100 μ L Optiphase 'Supermix' Liquid Scintillation cocktail reagent (Perkin Elmer) was added to the wells, followed by Scintillation counting (1450Microbeta Liquid Scintillation Counter, Wallac).

[00138] After removing the average background value for all data points, ki (app) data was calculated from nonlinear regression analysis of the initial rate data using the Prism Software package (GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San Diego California, USA).

While we have described a number of embodiments of this invention, it is apparent that our basic examples can be altered to provide other embodiments that utilize the compounds, methods and processes of this invention. It is, therefore, to be understood that the scope of the invention is to be limited by the claims rather than by the specific embodiments represented by the examples.

Claims (29)

1. A compound of formula I:

wherein

R¹Is H, C_1-6Aliphatic radicals or C_3-6A cycloaliphatic group;

g is-C (R)₂-or-O-;

l is C_0-3Aliphatic radical, optionally substituted by 0 to 3J^LSubstitution;

ring A is triazolyl, optionally substituted with 0-3J^ASubstitution;

ring B is a 5-6 membered aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S; ring B is optionally substituted with 0-5J^BSubstituted, and optionally fused, to ring B';

ring B' is a 5-8 membered aromatic or non-aromatic monocyclic ring containing 0-3 heteroatoms selected from O, N and S; ring B' is optionally substituted with 0-4J^B′Substitution;

each J^A、J^BAnd J^B′Independently is C_1-6Haloalkyl, halo, NO₂CN, Q or-Z-Q;

z is independently C_1-6Aliphatic radicals, optionally substituted by 0 to 3 occurrences of-NR-, -O-, -S-, -C (O) -, -C (═ NR) -, -C (═ NOR) -, -SO-or-SO₂-an interrupt; each Z is optionally substituted by 0-2J^ZSubstitution;

q is H; c_1-6An aliphatic group; a 3-8 membered aromatic or non-aromatic monocyclic ring having 0-3 heteroatoms independently selected from O, N and S; or an 8-12 membered aromatic or non-aromatic bicyclic ring system having 0-5 heteroatoms independently selected from O, N and S; each Q is optionally substituted by 0-5J^QSubstitution;

each J^LAnd J^ZIndependently is H, halo, C_1-6Aliphatic radical, C_3-6Cycloaliphatic radical, NO₂、CN、-NH₂、-NH(C_1-4Aliphatic group), -N (C)_1-4Aliphatic radical)₂、-OH、-O(C_1-4Aliphatic group), -CO₂H、-CO₂(C_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic group) or halo (C)_1-4Aliphatic groups);

each J^QIndependently is M or-Y-M;

each Y is independently unsubstituted C_1-6Aliphatic radicals, optionally substituted by 0 to 3 occurrences of-NR-, -O-, -S-, -C (O) -, -SO-or-SO₂-an interrupt;

each M is independently H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR′、SH、SR′、NH₂、NHR′、N(R′)₂、COH、COR′、CONH₂、CONHR′、CONR′₂、NHCOR′、NR′COR′、NHCONH₂、NHCONHR′、NHCON(R′)₂、SO₂NH₂、SO₂NHR′、SO₂N(R′)₂、NHSO₂R 'or NR' SO₂R′；

R is H or unsubstituted C_1-6An aliphatic group;

r' is unsubstituted C_1-6An aliphatic group; or two R' groups together with the atoms to which they are bonded form an unsubstituted 3-8 membered non-aromatic monocyclic ring having 0-1 heteroatoms independently selected from O, N and S.

2. The compound of claim 1, wherein G is-C (R)₂-。

3. The compound of claim 1, wherein G is O.

4. A compound according to any one of claims 1 to 3, wherein R¹Is H.

5. The compound of any one of claims 1-4, wherein ring a is

6. The compound of claim 5, wherein ring A is

7. A compound according to claim 5 or claim 6Wherein ring B is a 6-membered aromatic ring containing 0-2 nitrogen atoms, and ring B is optionally substituted with 0-5J^BAnd (4) substitution.

8. The compound of any one of claims 1-7, wherein ring B is fused to ring B', and ring B is optionally substituted with 0-5J^BSubstituted, ring B' is optionally substituted with 0-4J^B′And (4) substitution.

9. The compound of claim 9, wherein ring B 'is a 5-6 membered aromatic ring containing 0-3 heteroatoms selected from O, N and S, wherein ring B' is optionally substituted with 0-4J^B′And (4) substitution.

10. The compound of any one of claims 5-9, wherein J^AIs H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂R。

11. The compound of claim 10, wherein J^AIs H.

12. The compound of any one of claims 5-11, wherein J^BIs H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂R。

13. The compound of any one of claims 5-12, wherein J^B′Is H, C_1-6Aliphatic radical, C_3-6Cycloaliphatic, halo (C)_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic radical), C_3-6Heterocyclyl, halo, NO₂、CN、OH、OR、SH、SR、NH₂、NHR、N(R)₂、COH、COR、CONH₂、CONHR、CONR₂、NHCOR、NRCOR、NHCONH₂、NHCONHR、NHCON(R)₂、SO₂NH₂、SO₂NHR、SO₂N(R)₂、NHSO₂R or NRSO₂R。

14. A compound selected from the group consisting of:

15. a composition comprising a compound according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier, adjuvant or vehicle.

16. A method of inhibiting protein kinase activity in a patient comprising administering to said patient

a) The composition of claim 15; or

b) A compound according to any one of claims 1 to 14.

17. A method of inhibiting protein kinase activity in a biological sample comprising contacting said biological sample with:

a) the composition of claim 15; or

b) A compound according to any one of claims 1 to 14.

18. The method of claim 16 or claim 17, wherein the protein kinase is PLK.

19. The method of claim 18, wherein said protein kinase is Plk 1.

20. A method of treating a proliferative disorder, a neurodegenerative disorder, an autoimmune disorder, an inflammatory disorder, or an immunologically-mediated disorder in a patient, comprising administering to the patient:

a) the composition of claim 15; or

b) A compound according to any one of claims 1 to 14.

21. A method according to claim 16 or claim 20 comprising administering to the patient an additional therapeutic agent selected from the group consisting of: a chemotherapeutic or anti-proliferative agent, an anti-inflammatory agent, an immunomodulatory or immunosuppressive agent, a neurotrophic factor, a medicament for treating cardiovascular disease, a medicament for treating a destructive bone disorder, a medicament for treating a liver disease, an antiviral agent, a medicament for treating a blood disorder, a medicament for treating diabetes, or a medicament for treating an immunodeficiency disorder, wherein:

the additional therapeutic agent is appropriate with respect to the disease being treated; and

the additional therapeutic agent is administered with the composition as a single dosage form or separately from the composition as part of a multiple dosage form.

22. A method of treating melanoma, myeloma, leukemia, lymphoma, neuroblastoma, or a cancer selected from: colon, breast, stomach, ovary, cervix, lung, Central Nervous System (CNS), kidney, prostate, bladder or pancreas, wherein the method comprises administering to the patient

a) The composition of claim 15; or

b) A compound according to any one of claims 1 to 14.

23. A method of treating cancer in a patient, wherein the method comprises administering to the patient

a) The composition of claim 15; or

b) A compound according to any one of claims 1 to 14.

24. The method of claim 23, wherein said method comprises the step of disrupting mitosis in cancer cells by administering to said cells a therapeutic agent

a) The composition of claim 15; or

b) Compounds according to any of claims 1 to 14

Inhibition of PLK.

25. A process for preparing a compound of formula I:

wherein G is O, R¹Is H, ring A, ring B, J^A、J^BAnd L is as defined in any one of claims 1-14,

comprising reacting a compound of formula 3

Wherein G is O, ring A, ring B, J^A、J^BAnd L is as defined in any one of claims 1-14,

under suitable amide forming conditions to produce the compound of formula I.

26. A process for preparing a compound of formula 3:

wherein ring A, ring B, J^A、J^BAnd L is as defined in any one of claims 1 to 14, comprising reacting a compound of formula 2:

andcarrying out reaction;

wherein LG is a suitable leaving group, L, Ring B and J^BIs as defined in any one of claims 1 to 14;

under suitable O-C bond coupling conditions to produce the compound of formula 3.

27. The method of claim 26, further comprising the step of:

to a compound of formula 1:

addition ofWherein ring A is an aromatic ring containing a nitrogen atom capable of nucleophilic attack, J^AIs as defined in any one of claims 1 to 14;

the compound of formula 2 is produced via suitable conjugate addition conditions.

28. The method of claim 27, whereinIs a compound of formula 6:

29. the method of claim 28, further comprising the step of:

reacting a compound of formula 5:

treatment with an aldehyde and hydrazine generates a compound of formula 6.