HK1087700B

HK1087700B - Triazolopyridazines as protein kinases inhibitors

Info

Publication number: HK1087700B
Application number: HK06107864.2A
Authority: HK
Inventors: J.格林; R．小戈雷; A．C．皮尔斯
Original assignee: 沃泰克斯药物股份有限公司
Priority date: 2002-12-18
Filing date: 2003-12-17
Publication date: 2010-09-03

Description

Triazolopyridazines useful as protein kinase inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No.60/435,124 entitled "composition useful as a protein kinase inhibitor" filed on 12/18/2002, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to inhibitors of protein kinases. The invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various disorders.

Background

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.

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.the protein 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, Science 1991, 253, 407-23414; Hiles et al, Cell 1992, 70, 419-429; Kunz et al, Cell 1993, 73, 585-596; Garcia-Bustos et al, EMBO j.1994, 13, 2352-2361).

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 switchesAnd can regulate or modulate 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., osmotic 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.

Many diseases are associated with abnormal cellular responses triggered by the aforementioned protein kinase-mediated events. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, 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.

PIM-1 is a murine leukemia virus-activated proto-oncogene (Moloney murine leukemia virus provirus integration site) [ Cuypers, H.T., et al., Cell 37, 141-150(1984) ]. Expression of the protooncogene produces a 313 residue non-transmembrane serine/threonine kinase, including a kinase domain consisting of 253 amino acid residues. Two isoforms are known to be initiated by alternation (p44 and p33) [ Saris, c.j.m., et al, EMBO j., 10, 655-664(1991) ]. Two PIM-1 homologs have been described [ Baytel, d., Biochim biophysis Acta 1442, 274-85 (1998); feldman, J., et al, J Biol Chem 273, 16535-16543(1998) ]. At the amino acid level, PIM-2 and PIM-3 are 58% and 69% identical to PIM-1, respectively. PIM-1 is highly expressed in the liver and spleen during hematopoiesis, and expression is induced by cytokines such as GM-CSF, G-SCF, IL-3, IF- α, and IL-6[ Lilly, M., et al., Oncogene 7, 727-732 (1992); sato, n., et al, EMBO j.12, 4181-; jaster, r., et, Cell Signal 11, 331-; matikainen, S., et al, Blood 93, 1980-1991(1999) ].

PIM-1 has been implicated in lymphoma development. Induced PIM-1 expression and proto-oncogene c-myc synergistically increase the incidence of lymphoma [ Breuer m., et al, Nature 340, 61-63 (1989); van Lohuizen M., et al., Cell 65, 737-52(1991) ]. PIM-1 functions in the cytokine signaling pathway and has been shown to play a role in T cell development [ Schmidt, T., et al, EMBO J17, 5349-; jacobs, H., et al, JEM 190, 1059-. gp130 is a subunit common to receptors of the IL-6 cytokine family, and through its signaling activates the transcription factor STAT3, which is capable of causing proliferation of hematopoietic cells [ Hirano, T., et al., Oncogene 19, 2548-2556(2000) ]. Kinase activity PIM-1 appears to be essential for gp 130-mediated STAT3 proliferation signaling. In cooperation with c-myc, PIM-1 is able to promote STAT 3-mediated cell cycle progression and anti-apoptosis [ Shirogane, T., et al., Immunity 11, 709-719(1999) ]. PIM-1 also appears to be essential for IL-3-stimulated bone marrow-derived mast cell growth [ Domen J., et al, Blood 82, 1445-52(1993) ] and FDCP1 cell survival after IL-3 withdrawal [ Lilly, M., et al, Oncogene 18, 4022-4031(1999) ].

Furthermore, PIM-1 can control cell proliferation and survival by virtue of its phosphorylation of the well-defined cell cycle regulators cdc25[ Mochizuki, T., et al, J Biol Chem 274, 18659-.

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases consisting of a β -sheet rich amino-terminal leaf and a larger carboxy-terminal leaf, the latter being predominantly α -helical. CDKs exhibit 11 subdomains shared by all protein kinases with molecular masses ranging from 33 to 44 kD. This family of kinases, including CDK1, CDK2, CDK4 and CDK6, need to be phosphorylated at the residue corresponding to CDK-2 Thr160 in order to be fully active [ Meijer, l., Drug Resistance Updates, 3, 83-88(2000) ].

Each CDK complex is composed of regulatory cyclin subunits (e.g., cyclin A, B1, B2, D1, D2, D3, and E) and catalytic kinase subunits (e.g., CDK1, CDK-2, CDK4, CDK5, and CDK 6). Each distinct kinase/cyclin pair plays a role in regulating distinct specific phases of the cell cycle, referred to as G1, S, G2 and M phases [ Nigg, e., Nature Reviews, 2, 21-32 (2001); flatt, P., Pietenpol, J., Drug Metabolism Reviews, 32, 283- "305 (2000) ].

CDKs have been implicated in cell proliferative disorders, particularly cancer. Cell proliferation is the result of a direct or indirect deregulation of the cell differentiation cycle, and CDKs play a crucial role in the regulation of the various stages of this cycle. For example, overexpression of cyclin D1 is commonly associated with a number of human cancers, including breast, colon, hepatocellular, and gliomas [ Flatt, P., Pietenpol, J., Drug Metabolism Reviews, 32, 283-. The CDK-2/cyclin E complex plays a critical role in the progression of the cell cycle from the early G1 phase to the S phase, with overexpression of cyclin E being associated with a variety of solid tumors. Thus, inhibitors of cyclin D1, E or CDKs associated with them are useful targets for Cancer therapy [ Kaubisch, a., Schwartz, g., The Cancer Journal, 6, 192-212(2000) ].

CDKs, particularly CDK-2, also play a role in apoptosis and T-cell development. CDK-2 has been identified as a key regulator of thymocyte apoptosis [ Williams, O., etal, European Journal of Immunology, 709-. Stimulation of CDK-2 kinase activity is associated with the progression of apoptosis of thymocytes in response to specific stimuli. Inhibition of CDK-2 kinase activity may block this apoptosis, thereby protecting thymocytes.

In addition to regulating cell cycle and apoptosis, CDKs are also directly involved in the process of transcription. A large number of viruses require CDKs for their replication process. Examples of CDK inhibitors that prevent viral replication include human cytomegalovirus, herpes virus and varicella-zoster virus [ Meijer, l., Drug Resistance Updates, 3, 83-88(2000) ].

Inhibition of CDKs may also be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease. The appearance of Paired Helical Filaments (PHF) associated with Alzheimer's disease is caused by hyperphosphorylation of tau protein by CDK5/p25 [ Meijer, L., Drug resistance Updates, 3, 83-88(2000) ].

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, consisting of alpha and beta isoforms, each encoded by a different gene [ Coghlan et al, Chemistry & Biology, 7, 793-; kim and Kimmel, curr. opinion Genetics dev., 10, 508-. GSK-3 has been implicated in a variety of diseases, including diabetes, alzheimer's disease, CNS disorders such as manic-depressive psychotic disorders and neurodegenerative diseases, and cardiomyocyte hypertrophy [ PCT application No. wo 99/65897; WO 00/38675; haq et al, j.cell biol., 151, 117-30 (2000). These diseases are associated with abnormal operation of certain cellular signaling pathways in which GSK-3 plays a role. GSK-3 has been found to phosphorylate and modulate the activity of a number of regulatory proteins. These proteins include glycogen synthase, which is the rate-limiting enzyme necessary for glycogen synthesis, tau associated with microtubules, the gene transcription factor beta-catenin, the translation initiation factor e1F-2B, as well as ATP citrate lyase, shellac, heat shock factor-1, c-Jun, c-myc, c-myb, CREB and CEPB alpha. These different protein targets have led to GSK-3 involvement in many aspects of cellular metabolism, proliferation, differentiation and development.

In the GSK-3 mediated pathway associated with treatment of type II diabetes, insulin-induced signaling leads to cellular glucose uptake and glycogen synthesis. Along this pathway, GSK-3 is a negative regulator of insulin-induced signaling. Normally, the presence of insulin results in inhibition of GSK-3 mediated phosphorylation and inactivation of glycogen synthase. Inhibition of GSK-3 causes increased glycogen synthesis and glucose uptake [ Klein et al, PNAS, 93, 8455-9 (1996); cross et al, biochem.J., 303, 21-26 (1994); cohen, biochem. soc. trans., 21, 555-; and Massillon et al, biochem J.299, 123-128(1994) ]. However, in diabetic patients, if the insulin response is impaired, glycogen synthesis and glucose uptake cannot be increased despite the presence of relatively high blood insulin levels. This causes abnormally high blood glucose levels, the acute and long-term effects of which can ultimately lead to cardiovascular disease, renal failure and blindness. In such patients, normal insulin-induced inhibition of GSK-3 cannot occur. GSK-3 has also been reported to be overexpressed in I I diabetic patients [ see PCT application: WO 00/38675 ]. Therapeutic inhibitors of GSK-3 are therefore potentially useful in treating diabetic patients suffering from reduced insulin response.

GSK-3 activity is also associated with Alzheimer's disease. This disease is characterized by the well-known formation of β -amyloid peptides and intracellular neurofibrillary tangles. The a β peptide is derived from Amyloid Precursor Protein (APP), preceded by sequential proteolysis catalyzed by aspartyl protease BACE2, followed by presenilin-dependent γ -secretase cleavage. It has been demonstrated that anti-amyloid-plaque antibodies can delay cognitive decline in alzheimer's patients (hocket, Neuron, 2003, 38, 547-one 554), and thus other β -amyloid-lowering strategies (e.g. the development of drugs capable of inhibiting β -amyloid peptide) would be useful in the treatment of alzheimer's disease and other psychiatric and neurodegenerative diseases. In addition, neurofibrillary tangles contain hyperphosphorylated tau protein, in which tau is phosphorylated at abnormal sites, and thus drugs capable of inhibiting tau hyperphosphorylation would be useful in the treatment of Alzheimer's disease and other psychiatric and neurodegenerative diseases.

These abnormal sites in GSK-3 phosphorylated cells and animal models are known. In addition, GSK-3 has been shown to prevent hyperphosphorylation of τ in cells [ Lovestone et al, Current Biology 1994, 4, 1077-86; and Brown et al, Neuroreport 1997, 8, 3251-55. Thus, GSK-3 activity promotes the formation of neurofibrillary tangles and the progression of Alzheimer's disease. GSK-3 has also been shown to promote APP processing, and GSK-3 inhibitors (lithium) inhibit the production of A β peptides by inhibiting GSK-3 (Phietet al Nature 2003, 423, 435-. Thus, the development of GSK-3 inhibitors would be useful in reducing the formation of amyloid plaques and neurofibrillary tangles, which are pathological hallmarks of alzheimer's disease, and would also be useful in the treatment of other psychiatric and neurodegenerative diseases.

Another GSK-3 substrate is β -catenin, which is degraded by GSK-3 after phosphorylation. Reduction of β -catenin levels has been reported in schizophrenic patients, and has also been associated with other diseases involving increased neuronal cell death [ Zhong et al, Nature, 395, 698-702 (1998); takashima et al, PNAS, 90, 7789-93 (1993); andPei et al, j.neuro, make pathol.exp, 56, 70-78 (1997).

GSK-3 activity is also associated with stroke [ Wang et al, Brain Res, 859, 381-5 (2000); sasaki et al, Neurol Res, 23, 588-92 (2001); hashimoto et al, J.biol.chem, 2002, 277, 32985-. Another particularly relevant family of protein kinases are the Src family of kinases. These kinases have been implicated in cancer, immune system dysfunction and bone remodeling diseases. For general comments, see Thomas andcrugge, annu.rev.cell dev.biol. (1997)13, 513; lawrence and ni, pharmacol. ther. (1998)77, 81; tatosian and Mizenia, biochemistry (Moscow) 2000, 65, 49; boschelli et al, drug of the Future 2000, 25(7), 717, (2000).

In mammals, Src family members include the following eight kinases: src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk. They are receptor-free protein kinases with molecular weights from 52 to 62 kD. All are characterized by a common structural organization, which consists of six different functional domains: src homology domain 4(SH4), a unique domain, SH3 domain, SH2 domain, a catalytic domain (SH1) and C-terminal regulatory region. Tatosian et al biochemistry (Moscow)65, 49-58 (2000).

Based on published studies, Src kinase is considered as a potential therapeutic target for a variety of human diseases. Mice deficient in Src develop osteopetrosis or bone formation because osteoclasts inhibit bone resorption. This suggests that Src inhibition can treat osteoporosis caused by abnormally high bone resorption. Soriano et al, Cell, 69, 551(1992) and Soriano et al, Cell, 64, 693 (1991).

Inhibition of inflammatory bone destruction in joints has been achieved by virtue of overexpression of CSK in rheumatoid synoviocytes and osteoclasts. Takayanagi et al, j.clin.invest, 104, 137 (1999). CSK or C-terminal Src kinase phosphorylate, thereby inhibiting the catalytic activity of Src. This suggests that Src inhibition can prevent joint destruction specific to rheumatoid arthritis patients. Boschelli et al, Drugs of the Future 2000, 25(7), 717, (2000).

Src also plays a role in the replication of hepatitis b virus. In one step required for virus propagation, the virally encoded transcription factor HBx activates Src. Klein et al, EMBO j., 18, 5019, (1999) and Klein et al, mol.

A number of studies have linked Src expression to cancers such as colon, breast, liver and pancreatic cancers, certain B-cell leukemias and lymphomas. Talamonti et al, j.clin.invest, 91, 53 (1993); lutz et al, biochem. biophysis. res.243, 503 (1998); rosen et al, j.biol.chem., 261, 13754 (1986); bolen et al, proc.natl.acad.sci.usa, 84, 2251 (1987); masakiet al, Hepatology, 27, 1257 (1998); bisscardi et al, adv. cancer res, 76, 61 (1999); lynch et al, Leukemia, 7, 1416 (1993). In addition, antisense Src expressed in ovarian and colon tumor cells has been shown to inhibit tumor growth. Wiener et al, clin. cancer res, 5, 2164 (1999); staley et al, Cell Growth Diff, 8, 269 (1997).

Other Src family kinases are also potential therapeutic targets. Lck plays a role in T-cell signaling. Mice lacking the Lck gene have poor thymocyte developmental competence. The function of Lck as a positive activator of T-cell signaling suggests that Lck inhibitors may be useful in the treatment of autoimmune diseases, such as rheumatoid arthritis. Molina et al, Nature, 357, 161 (1992). Hck, Fgr and Lyn have been identified as important mediators of integrin signaling in bone marrow leukocytes. Lowell et al, j.leukoc.biol., 65, 313 (1999). Inhibition of these kinase mediators is therefore useful in the treatment of inflammation. Boschellie et al, drug of the Future 2000, 25(7), 717, (2000).

Thus, in view of the inadequate treatment of most of the following conditions currently available, there is a great need to develop inhibitors of PIM-1, CDK-2, SRC and GSK-3 protein kinases that can be used to treat a variety of diseases or conditions associated with PIM-1, CDK-2, SRC or GSK-3 activation.

Disclosure of Invention

It has now been found that the compounds of the present invention, and pharmaceutically acceptable compositions thereof, are effective as inhibitors of PIM-1, CDK-2, SRC and GSK-3 protein kinases. In certain other embodiments, these compounds are effective as inhibitors of PIM-1 protein kinase. These compounds have the general formula I:

or a pharmaceutically acceptable derivative thereof, wherein R¹And R²Is defined as follows.

These compounds and pharmaceutical compositions thereof are useful for treating or preventing a variety of conditions including, but not limited to, heart disease, diabetes, alzheimer's disease, immunodeficiency disorders, inflammatory diseases, allergic diseases, autoimmune diseases, destructive bone disorders (e.g., osteoporosis), proliferative diseases, infectious diseases, immunologically-mediated diseases, and viral diseases. The compositions are also useful in methods of preventing cell death and proliferation, and thus can be used to treat or prevent reperfusion/ischemia in stroke, heart attack, and organ hypoxia. The compositions may also be used in methods of preventing thrombin-induced platelet aggregation. The compositions are particularly useful in conditions such as Chronic Myelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML), Acute Promyelocytic Leukemia (APL), rheumatoid arthritis, asthma, osteoarthritis, ischemia, cancer, liver diseases including hepatic ischemia, heart diseases such as myocardial infarction and congestive heart failure, pathological immune conditions involving T cell activation, and neurodegenerative diseases.

Detailed description of the invention

1. General description of the compounds of the invention:

the present invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof,

wherein R is¹Is OR³、SR³Or NR³R⁴(ii) a Wherein R is³And R⁴Independently for each occurrence of (U)_mR', wherein U is optionally substituted C_1-6An alkylene chain wherein up to two methylene units of the chain are optionally and independently substituted by-C (O) -, -C (O) -, -CONR-, -CONRNR-, -CO₂-、-OC(O)-、-NRCO₂-、-O-、-NRCONR-、-OC(O)NR-、-NRNR、-NRCO-、-S-、-SO-、-SO₂-、-NR-、-SO₂NR-or-NRSO₂-a stationReplacing; m is 0 or 1; or wherein R is³And R⁴Together with the nitrogen, an optionally substituted 5-8 membered heterocyclyl or heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

each occurrence of R is independently selected from hydrogen or optionally substituted C_1-6An aliphatic radical, each occurrence of R' being independently selected from hydrogen or an optionally substituted radical selected from C_1-8Aliphatic radical, C_6-10An aryl, heteroaryl ring having 5 to 10 ring atoms or a heterocyclyl ring having 3 to 10 ring atoms, or wherein R and R ', taken together or R' taken twice on the same or different substituents, together form a 5-8 membered heterocyclyl or heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

R²is- (T)_nAr¹Wherein T is NR; n is 0 or 1; ar (Ar)¹Is a 3-7 membered saturated, partially unsaturated or fully unsaturated monocyclic ring having 0-3 heteroatoms independently selected from nitrogen, oxygen or sulfur, or an 8-10 membered saturated, partially unsaturated or fully unsaturated bicyclic ring system having 0-5 heteroatoms independently selected from nitrogen, oxygen or sulfur.

In certain embodiments, the compounds of formula I exclude one or more or all of the following compounds:

1) if R is²Is an optionally substituted 1, 3, 5-triazine, then R¹Is not an N-morpholino group;

2) if R is²Is nitro-substituted pyrazolyl, furyl or thiophene, then R¹Is not NR³R⁴；

3) If R is²Is furyl, then R²Is not NH₂；

4) If R is²Is optionally substituted pyridyl or phenyl, then R¹Is not OR³Wherein R is³Is halogen substituted alkyl;

5) if R is²Is substituted by haloalkyl or haloalkoxyPhenyl of (e), then R¹Is not NHC_1-4Alkyl or O (CH)₂)₂N(Me)₂；

6) Compounds of formula I exclude:

a. butyric acid, 2- (benzylamino) -3- [ (3-phenyl-1, 2, 4-triazolo [4, 3-b ] pyridazin-6-yl) hydrazono ] -methyl ester;

b. benzamide, N- [2, 5-dihydro-3-methyl-5-oxo-1- (3-phenyl-1, 2, 4-triazolo [4, 3-b ] pyridazin-6-yl) -1H-pyrazol-4-yl ]; and

2-propenoic acid, 2- (benzylamino) -3- [3, 5-dimethyl-1- (3-phenyl-1, 2, 4-triazolo [4, 3-b ] pyridazin-6-yl) -1H-pyrazol-4-yl ];

7) if R is²Is one or more occurrences of OMe, Me, NO₂Cl or CF₃Substituted phenyl, then R¹Morpholino or piperazinyl which is not optionally substituted;

8) if R is²Is phenyl or fluorine-substituted phenyl, then R¹Is not-O-CH₂- (triazolyl);

9) if R is¹is-NH (cyclopropyl), then R²CF not appearing once in para position₃Substituted phenyl;

10) if R is²Is unsubstituted phenyl, then R¹Is not-SR³Wherein R is³Is in the meta position by CF₃Substituted phenyl, twice occurring OCH₃、(CH₂)₂OH、-(CH₂)COOCH₂CH₃Substituted phenyl or phenyl substituted in para position with one occurrence of Cl; or

11) If R is²Is unsubstituted phenyl, then R¹Is not nh (ch) ═ NOH.

2. Compounds and definitions:

the compounds of the present invention include those generally described above, further illustrated as major classes, minor classes, and species disclosed herein. Unless otherwise indicated, the following definitions will apply. For the purposes of the present invention, chemical elements will be identified according to the CAS version of Handbook of chemistry and Physics, 75th Ed of the periodic Table of elements. 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", 5th Ed., Ed.: smith, m.b. and March, j., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

As described herein, the compounds of the invention may be optionally substituted with one or more substituents, such as those set forth in the summary above, or as exemplified by particular classes, subclasses, and species of the invention. It is to be understood that the phrase "optionally substituted" is used interchangeably with the phrase "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. 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.

As used herein, the term "aliphatic" or "aliphatic radical" refers to a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or that contains one or more carbon atomsAn unsaturated unit, alternatively a monocyclic or bicyclic hydrocarbon, is fully saturated or contains one or more unsaturated units, but is not aromatic (also referred to herein as a "carbocycle", "cycloaliphatic" or "cycloalkyl"), and 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. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") denotes 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, which has a single point of attachment to the rest of the molecule, wherein any single ring in said bicyclic ring system is a 3-7 membered ring. Suitable aliphatic groups include, but are not limited to, linear or branched substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl, or (cycloalkyl) alkenyl.

The term "heteroaliphatic" as used herein, refers to an aliphatic group in which one or two carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon. Heteroaliphatic groups may be substituted or unsubstituted, straight or branched chain, cyclic or acyclic, and include "heterocyclic", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" groups.

The term "heterocycle", "heterocyclyl", "heterocycloaliphatic", or "heterocyclic" as used herein, refers to a non-aromatic, monocyclic, bicyclic, or tricyclic ring system in which one or more ring members are independently selected heteroatoms. In some embodiments, "heterocycle", "heterocyclyl", or "heterocyclic group,

A "heterocycloaliphatic" 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.

The term "heteroatom" means one or more oxygen, sulfur, nitrogen, phosphorus or silicon (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)).

The term "unsaturated" as used herein means that the moiety has one or more units of unsaturation.

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.

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 term "halogen" denotes F, Cl, Br or I.

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 a heteroaryl ring system as defined below.

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".

Aryl (including aralkyl, aralkoxy, aryloxyalkyl, and the like) or heteroaryl (including heteroaralkyl and heteroaralkoxy, and the like) may contain one or more substituents and thus may be "optionally substituted". In the above and in the present invention, unless otherwise defined, suitable substituents on the unsaturated carbon atoms of aryl or heteroaryl groups are generally selected from halogen, -R^o、-OR^o、-SR^oOptionally by R^oSubstituted phenyl (Ph), optionally substituted with R^osubstituted-O (Ph), optionally substituted with R^oSubstituted- (CH)₂)_1-2(Ph), optionally substituted by R^osubstituted-CH ═ CH (Ph), -NO₂、-CN、-N(R^o)₂、-NR^oC(O)R^o、-NR^oC(S)R^o、-NR^oC(O)N(R^o)₂、-NR^oC(S)N(R^o)₂、-NR^oCO₂R^o、-NR^oNR^oC(O)R^o、-NR^oNR^oC(O)N(R^o)₂、-NR^oNR^oCO₂R^o、-C(O)C(O)R^o、-C(O)CH₂C(O)R^o、-CO₂R^o、-C(O)R^o、-C(S)R^o、-C(O)N(R^o)₂、-C(S)N(R^o)₂、-OC(O)N(R^o)₂、-OC(O)R^o、-C(O)N(OR^o)R^o、-CN(OR^o)R^o、-S(O)₂R^o、-S(O)₃R^o、-SO₂N(R^o)₂、-S(O)R^o、-NR^oSO₂N(R^o)₂、-NR^oSO₂R^o、-N(OR^o)R^o、-C(＝NH)-N(R^o)₂、-P(O)₂R^o、-PO(R^o)₂、-OPO(R^o)₂、-(CH₂)_0-2NHC(O)R^oOptionally by R^oSubstituted phenyl (Ph), optionally substituted with R^osubstituted-O (Ph), optionally substituted with R^oSubstituted- (CH)₂)_1-2(Ph), or optionally R^osubstituted-CH ═ CH (ph); wherein each independently occurring R^oSelected from hydrogen, optionally substituted C_1-6An aliphatic radical, an unsubstituted 5-6 membered heteroaryl or heterocycle, phenyl (Ph), -O (Ph), or-CH₂(Ph), or, although as defined above, two independent occurrences of R on the same substituent or different substituents^oAnd each R^oThe atoms to which the groups are bonded together form an optionally substituted 3-12 membered saturated, partially unsaturated or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

R^oIs selected from NH₂、NH(C_1-4Aliphatic radical), N (C)_1-4Aliphatic radical)₂Halogen, C_1-4Aliphatic radical, OH, O- (C)_1-4Aliphatic group), NO₂、CN、CO₂H、CO₂(C_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic group) or halogeno C_1-4Aliphatic radical, wherein R^oEach of the above C_1-4Aliphatic groups are unsubstituted.

An aliphatic or heteroaliphatic group or a non-aromatic heterocycle may contain one or more substituents and thus may be "optionally substituted". Unless otherwise defined above and herein, suitable substituents on saturated carbon atoms of aliphatic or heteroaliphatic groups or non-aromatic heterocycles are selected from those listed above for aryl or heteroaryl unsaturated carbons, and additionally include the following groups: two (R) ═ O, ═ S, ═ NNHR, ═ NN (R)₂、＝NNHC(O)R^*、＝NNHCO₂(alkyl) ═ NNHSO₂(alkyl) or ═ NR^*Wherein each R is^*Independently selected from hydrogen or optionally substituted C_1-6An aliphatic group.

Unless otherwise defined above and herein, optional substituents on non-aromatic heterocyclic nitrogen are generally selected from the group consisting of-R⁺、-N(R⁺)₂、-C(O)R⁺、-CO₂R⁺、-C(O)C(O)R⁺、-C(O)CH₂C(O)R⁺、-SO₂R⁺、-SO₂N(R⁺)₂、-C(＝S)N(R⁺)₂、-C(＝NH)-N(R⁺)₂or-NR⁺SO₂R⁺(ii) a Wherein R is⁺Is hydrogen, optionally substituted C_1-6An aliphatic group, an optionally substituted phenyl group (Ph), an optionally substituted-O (Ph), an optionally substituted-CH₂(Ph), optionally substituted- (CH)₂)_1-2(Ph), optionally substituted-CH ═ CH (Ph), or unsubstituted 5-6 membered heteroaryl or heterocyclic ring having one to four heteroatoms independently selected from oxygen, nitrogen or sulfur, or two independent occurrences of R, although as defined above, on the same substituent or on different substituents⁺And each R⁺The atoms to which the groups are bonded together form an optionally substituted 3-12 membered saturated, partially unsaturated or fully unsaturated monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

R⁺Is selected from-NH or an optional substituent on the phenyl ring₂、-NH(C_1-4Aliphatic radical), -N (C)_1-4Aliphatic radical)₂Halogen, C1_-4Aliphatic radical, -OH, -O- (C)_1-4Aliphatic radical), -NO₂、-CN、-CO₂H、-CO₂(C_1-4Aliphatic group), -O (halogeno-C)_1-4Aliphatic group) or halogeno C_1-4Aliphatic radical, wherein R⁺Each of the above C_1-4Aliphatic groups are unsubstituted.

The term "alkylene chain" denotes a straight or branched carbon chain, which may be fully saturated or have one or more units of unsaturation, and has two points of attachment to the rest of the molecule.

As noted above, in some embodiments, two independent occurrences of R^o(or R)⁺R, R' or any other similarly defined variable herein) together with the atoms to which they are bonded form an optionally substituted 3-12 membered saturated, partially unsaturated, or fully unsaturated mono-or mono-unsaturated ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfurA ring or a bicyclic ring.

Two (one) independently occurring R^o(or R)⁺R, R', or any other variable similarly defined herein) along with the atom to which each variable is bonded include, but are not limited to, the following: a) two independently occurring R^o(or R)⁺R, R' or any other variable similarly defined herein) to the same atom and together with that atom form a ring, e.g., N (R)^o)₂In which two R are present^oTogether with the nitrogen atom, form piperidin-1-yl, piperazin-1-yl, or morpholin-4-yl; and b) two independently occurring R^o(or R)⁺R, R' or any other variable similarly defined herein) to different atoms and together with these atoms form a ring, e.g.OR in which the phenyl radical is present twice^oSubstitution, R of both occurrences^oTogether with the oxygen atoms to which they are bonded form a fused 6-membered oxygen containing ring:

it is understood that two independent occurrences of R^o(or R)⁺R, R', or any other variable similarly defined herein) along with the atoms to which each variable is bonded may form a variety of other rings, and the above detailed examples are not intended to be limiting. 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, and geometric (or conformational) mixtures of these compounds are within the scope of the invention.

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

3. Description of exemplary Compounds:

in certain embodiments, R is as described generally above¹Is NR³R⁴、OR³Or SR³The compound has one of the following general formulas II, III or IV:

in a preferred embodiment, n is 1, T is NR, R²is-NRAr¹The compound has one of the general formulae IIA, IIIA or IVA:

in certain other preferred embodiments, m is 0 and R is²is-Ar¹The compound has one of the general formulae IIB, IIIB or IVB:

in certain embodiments, R⁴Is hydrogen or optionally substituted C_1-4Alkyl radical, R³Is an optionally substituted aryl, heteroaryl, cycloaliphatic or heterocycloaliphatic group. In certain other embodiments, R³Is an optionally substituted 5-or 6-membered aryl or heteroaryl group. In other embodiments, R³Is an optionally substituted 3-7 membered cycloaliphatic or heterocycloaliphatic group.

In certain embodiments, R⁴Is hydrogen or optionally substituted C_1-4Alkyl radical, R³Is an optionally substituted cyclic group selected from:

wherein any substitutable carbon or nitrogen atom is optionally substituted, wherein y is 0-5, Z is a bond or C_1-6Alkylene chain, wherein up to two non-adjacent methylene units of Q are optionally substituted by CO, CO₂、COCO、CONR、OCONR、NRNR、NRNRCO、NRCO、NRCO₂、NRCONR、SO、SO₂、NRSO₂、SO₂NR、NRSO₂NR, O, S or NR; r^YIndependently for each occurrence of (A) is selected from R', halogen, NO₂、CN、OR’、SR’、N(R’)₂、NR’C(O)R’、NR’C(O)N(R’)₂、NR’CO₂R’、C(O)R’、CO₂R’、OC(O)R’、C(O)N(R’)₂、OC(O)N(R’)₂、SOR’、SO₂R’、SO₂N(R’)₂、NR’SO₂R’、NR’SO₂N(R’)₂C (O) R' or C (O) CH₂C(O)R’。

In certain other embodiments, R³And R⁴Together with the nitrogen atom, form a group selected from:

in a more preferred embodiment, R³Selected from one of the following groups:

in certain preferred embodiments, y is 0-3, R³By 0-3 occurrences of ZR^YAnd (4) substitution. In certain other preferred embodiments, y is 1 or 2. In other preferred embodiments, y is 0 and R³Is unsubstituted.

In a preferred embodiment, ZR^YIndependently for each occurrence of (A) is halogen, CN, NO₂Or an optionally substituted group selected from C_1-4Alkyl, aryl, aralkyl, -N (R')₂、CH₂N(R’)₂、-OR’、CH₂OR’、-SR’、CH₂SR ', COOR' or-S (O)₂N(R’)₂. In a more preferred embodiment, ZR^YEach occurrence of (A) is independently Cl, Br, F, CN, CF₃、COOH、-N(CH₃)₂、-OH、CH₂OH or an optionally substituted group selected from C_1-4Alkoxy radical, C_1-4Alkyl, phenyl, phenoxy, benzyl or benzyloxy. Most preferred ZR^YGroups include those shown in table 1 below.

In other embodiments, R⁴Is hydrogen or optionally substituted C_1-4Alkyl radical, R³Is (U)_mR' wherein m is 1 and U is optionally substituted C_1-6An alkylene chain wherein up to two methylene units of the chain are optionally and independently substituted by-C (O) -, -C (O) -, -CONR-, -CONRNR-, -CO₂-、-OC(O)-、-NRCO₂-、-O-、-NRCONR-、-O(CO)NR’-、-NRNR、-NRCO-、-S-、-SO-、-SO₂-、-NR-、-SO₂NR-or-NRSO₂-substituted. In certain preferred embodiments, U is optionally substituted C_1-4An alkylene chain, wherein one methylene unit of the chain is optionally substituted by-C (O) -, -CONR-, -CO₂-, -OC (O) -, -O-or-NRCO-. In a more preferred embodiment, U is-CH₂(C＝O)NH-、-CH₂(C＝O)O-、-(CH₂)₂O-or-CH ═ NO-, wherein each occurrence of R' is independently hydrogen or C_1-4An alkyl group.

As generally described above, R²Is (T)_nAr¹Wherein n is 0 or 1 and T is NR. Preferred Ar is¹Groups include optionally substituted cyclic groups selected from:

wherein any substitutable carbon or nitrogen atom is optionally substituted, wherein x is 0 to 5, Q is a bond or C_1-6Alkylene chain, wherein up to two non-adjacent methylene units of Q are optionally substituted by CO, CO₂、COCO、CONR、OCONR、NRNR、NRNRCO、NRCO、NRCO₂、NRCONR、SO、SO₂、NRSO₂、SO₂NR、NRSO₂NR, O, S or NR; r^XIndependently for each occurrence of (A) is selected from R', halogen, NO₂、CN、OR’、SR’、N(R’)₂、NR’C(O)R’、NR’C(O)N(R’)₂、NR’CO₂R’、C(O)R’、CO₂R’、OC(O)R’、C(O)N(R’)₂、OC(O)N(R’)₂、SOR’、SO₂R’、SO₂N(R’)₂、NR’SO₂R’、NR’SO₂N(R’)₂C (O) R' or C (O) CH₂C(O)R’。

In certain preferred embodiments, x is 0-3, R²QR appeared 0-3 times^XAnd (4) substitution. In certain other preferred embodiments, x is 1 or 2. In other preferred embodiments, x is 0 and R is²Is unsubstituted.

In a preferred embodiment, QR^XIndependently for each occurrence of (A) is halogen, CN, NO₂Or an optionally substituted group selected from C_1-4Alkyl, aryl, aralkyl, -N (R')₂、CH₂N(R’)₂、-OR’、CH₂OR’、-SR’、CH₂SR ', COOR' or-S (O)₂N(R’)₂. In a more preferred embodiment, QR^XEach occurrence of (A) is independently Cl, Br, F, CN, CF₃、COOH、-N(CH₃)₂、-OH、CH₂OH or an optionally substituted group selected from C_1-4Alkoxy radical, C_1-4Alkyl, phenyl, phenoxy, benzyl or benzyloxy. Most preferred QR^XGroups include those shown in table 1 below.

It will be appreciated that, with respect to the above compounds, certain other compounds are of particular interest. For example, in certain exemplary embodiments, with respect to the compounds of formulae IIA, IIIA and IVA described above, particularly relevant compounds include those wherein R is⁴Is hydrogen or C_1-4An alkyl group; r³Is optionally substituted phenyl; r²is-NRAr¹These compounds have one of the following formulae:

in certain other preferred embodiments, R²is-Ar¹These compounds have one of the following general formulae:

in certain other exemplary embodiments, for compounds of formulas II, III, and IV described above, R²Is NRAr¹，Ar¹Is an optionally substituted phenyl group, these compounds having one of the following formulae:

in certain other preferred embodiments, R²Is Ar¹Wherein Ar is¹Is an optionally substituted phenyl group, these compounds having one of the following formulae:

it will be appreciated that certain subclasses of the above compounds are of particular interest.

For example, in certain preferred embodiments, certain substituents of the compounds as described above generally by formulas II-A-a, III-A-a, IV-A-a, II-B-a, III-B-a, and IV-B-a are defined as follows:

a.R⁴is hydrogen or C_1-4An alkyl group;

b. if n is 1 and T is NR, then R is hydrogen or C_1-4An alkyl group;

c.R³is selected from

Wherein y is 0-3 and Z is a bond or C_1-6Alkylene chain, wherein up to two non-adjacent methylene units of Q are optionally substituted by CO, CO₂、COCO、CONR、OCONR、NRNR、NRNRCO、NRCO、NRCO₂、NRCONR、SO、SO₂、NRSO₂、SO₂NR、NRSO₂NR, O, S or NR; r^YIndependently for each occurrence of (A) is selected from R', halogen, NO₂、CN、OR’、SR’、N(R’)₂、NR’C(O)R’、NR’C(O)N(R’)₂、NR’CO₂R’、C(O)R’、CO₂R’、OC(O)R’、C(O)N(R’)₂、OC(O)N(R’)₂、SOR’、SO₂R’、SO₂N(R’)₂、NR’SO₂R’、NR’SO₂N(R’)₂C (O) R' or C (O) CH₂C(O)R’；

d.x is 0-3, Q is a bond or C_1-6Alkylene chain, wherein up to two non-adjacent methylene units of Q are optionally substituted by CO, CO₂、COCO、CONR、OCONR、NRNR、NRNRCO、NRCO、NRCO₂、NRCONR、SO、SO₂、NRSO₂、SO₂NR、NRSO₂NR, O, S or NR; r^XIndependently for each occurrence of (A) is selected from R', halogen, NO₂、CN、OR’、SR’、N(R’)₂、NR’C(O)R’、NR’C(O)N(R’)₂、NR’CO₂R’、C(O)R’、CO₂R’、OC(O)R’、C(O)N(R’)₂、OC(O)N(R’)₂、SOR’、SO₂R’、SO₂N(R’)₂、NR’SO₂R’、NR’SO₂N(R’)₂C (O) R' or C (O) CH₂C(O)R’。

In certain preferred embodiments, with respect to the above compounds, y is 0-3, and R is³By 0-3 occurrences of ZR^YAnd (4) substitution. In certain other preferred embodiments, y is 1 or 2. In other preferred embodiments, y is 0 and R³Is unsubstituted.

In certain preferred embodiments, with respect to the above compounds, x is 0-3 and R is²QR appeared 0-3 times^XAnd (4) substitution. In certain other preferred embodiments, x is 1 or 2. In other preferred embodiments, x is 0 and R is²Is unsubstituted.

In other exemplary embodiments, for compounds of formula II, III or IV, R³Is an optionally substituted radical selected from phenyl, cyclohexyl, cyclobutyl or cyclopropyl, R²Is NRAr¹Wherein Ar is¹Is an optionally substituted phenyl group, these compounds having one of the following formulae:

in other exemplary embodiments, for compounds of formula II, III or IV, R³Is an optionally substituted radical selected from phenyl, cyclohexyl, cyclobutyl or cyclopropyl, R²Is Ar¹Wherein Ar is¹Is an optionally substituted phenyl group, these compounds having one of the following formulae:

For example, in certain preferred embodiments, certain substituents of the above compounds are defined as follows:

a.R⁴is hydrogen or C_1-4An alkyl group;

b. if n is 1 and T is NR, then R is hydrogen or C_1-4An alkyl group;

c.y is 0-3, Z is a bond or C_1-6Alkylene chain, wherein up to two non-adjacent methylene units of Q are optionally substituted by CO, CO₂、COCO、CONR、OCONR、NRNR、NRNRCO、NRCO、NRCO₂、NRCONR、SO、SO₂、NRSO₂、SO₂NR、NRSO₂NR, O, S or NR; r^YIndependently for each occurrence of (A) is selected from R', halogen, NO₂、CN、OR’、SR’、N(R’)₂、NR’C(O)R’、NR’C(O)N(R’)₂、NR’CO₂R’、C(O)R’、CO₂R’、OC(O)R’、C(O)N(R’)₂、OC(O)N(R’)₂、SOR’、SO₂R’、SO₂N(R’)₂、NR’SO₂R’、NR’SO₂N(R’)₂C (O) R' or C (O) CH₂C(O)R’；

Representative examples of compounds of formula I are described in table 1 below.

Table 1: examples of Compounds of formula I

4. General synthetic methods:

the compounds of the present invention can generally be prepared by methods known to those skilled in the art for the preparation of analogous compounds, as illustrated in the following general schemes and preparations below.

Scheme 1

Scheme 1 above shows a general procedure for the preparation of compounds of formula III'. For example, the compounds of the invention can be prepared by reacting the starting materials (A) with POCl₃And reacting to obtain chloride (II'). (II ') with an appropriate amine to give the desired compound of formula (III').

Scheme 2 below depicts the synthesis of certain exemplary compounds, wherein R²Is phenyl, these compounds are also prepared according to the general procedures described above.

Scheme 2

Scheme 3 below depicts the synthesis of certain exemplary compounds, wherein R²Is phenyl, R¹Is NHR³Wherein R is³Is (CH)₂) (C ═ O) OH, these compounds also being prepared according to the general procedure described above.

Scheme 3

Scheme 4 below depicts the synthesis of certain exemplary compounds, wherein R²Is (T)_nAr¹N is 1, T is NH, Ar¹Are optionally substituted phenyl groups, and these compounds are also prepared according to the general procedures described above.

Scheme 4

While certain exemplary embodiments are depicted and described above and herein, it will be appreciated that the compounds of the invention can be prepared by the methods generally described above using appropriate starting materials.

5. Use, formulation and administration:

pharmaceutically acceptable compositions

As discussed above, the present invention provides compounds that are inhibitors of protein kinases and thus are useful for treating diseases, disorders, and conditions including, but not limited to, proliferative diseases, cardiac diseases, neurodegenerative diseases, psychiatric disorders, autoimmune diseases, conditions associated with organ transplantation, inflammatory diseases, immunologically mediated diseases, viral diseases, or bone diseases. In preferred embodiments, the compounds are useful for the treatment of allergy, asthma, diabetes, Alzheimer's disease, Huntington's disease, Parkinson's disease, dementia associated with AIDS, amyotrophic lateral sclerosis (AML, Lou Gehrig's disease), Multiple Sclerosis (MS), schizophrenia, cardiomyocyte hypertrophy, reperfusion/ischemia (e.g., stroke), hair loss, cancer, hepatomegaly, cardiovascular disease (including cardiac hypertrophy), cystic fibrosis, viral diseases, autoimmune diseases, atherosclerosis, restenosis, psoriasis, inflammation, hypertension, angina pectoris, cerebral vasoconstriction, peripheral circulation disorders, premature labor, arteriosclerosis, vasospasm (cerebral vasospasm, coronary vasospasm), retinopathy, Erectile Dysfunction (ED), AIDS, osteoporosis, Crohn's disease and colitis, axonal warts and Reynaud's disease. In a preferred embodiment, the disease, condition or disorder is atherosclerosis, hypertension, Erectile Dysfunction (ED), reperfusion/ischemia (e.g., stroke), or vasospasm (cerebral vasospasm and coronary vasospasm).

Thus, in another aspect of the invention, pharmaceutically acceptable compositions are provided, wherein these compositions comprise any of the compounds as 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.

It will also be appreciated that certain compounds of the invention can be present in free form for use in therapy, or as appropriate pharmaceutically acceptable derivatives thereof. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adduct or derivative that upon administration to a patient in need thereof is capable of providing, directly or indirectly, a compound as described herein or a metabolite or residue thereof. The term "pharmaceutically acceptable salt" as used herein, means those salts 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. "pharmaceutically acceptable salt" means any non-toxic salt or ester salt of a compound of the present invention which, upon administration to a recipient, is capable of providing, directly or indirectly, a compound of the present invention or an inhibitorily active metabolite or residue thereof. The term "its inhibitory active metabolite or residue" as used herein means that its metabolite or residue is also an inhibitor of PIM-1, CDK-2, SRC or GSK-3.

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. 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, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, embonate, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, 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. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Other pharmaceutically acceptable salts include, when 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.

As noted above, the pharmaceutically acceptable compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, adjuvant or vehicle, as described herein, including any and all solvents, diluents or other liquid excipients, dispersing or suspending 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, SixteenthEdition, e.w. martin (Mack Publishing co., Easton, Pa., 1980) disclose various carriers for formulating pharmaceutically acceptable compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, e.g., any other component that produces any undesirable biological effect or interacts in a deleterious manner with a pharmaceutically acceptable composition, its use is contemplated as falling within the scope of the present invention. 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.

Use of compounds and pharmaceutically acceptable compositions

In another aspect, there is provided a method of treating or lessening the severity of a proliferative disease, a cardiac disease, a neurodegenerative disease, a psychiatric disorder, an autoimmune disease, a condition associated with organ transplantation, an inflammatory disease, an immunologically-mediated disease, a viral disease or a bone disease comprising administering to a patient in need of such treatment an effective amount of the compound or a pharmaceutically acceptable composition comprising the compound. In certain embodiments of the invention, an "effective amount" of a compound or pharmaceutically acceptable composition is an amount effective to treat or reduce the severity of a proliferative disease, a cardiac disease, a neurodegenerative disease, a psychiatric disorder, an autoimmune disease, a condition associated with organ transplantation, an inflammatory disease, an immunologically-mediated disease, a viral disease or a bone disease. The compounds and compositions according to the methods of the present invention can be administered in any amount and by any route of administration that is effective for treating or lessening the severity of a proliferative disease, a cardiac disease, a neurodegenerative disease, a psychiatric disorder, an autoimmune disease, a condition associated with organ transplantation, an inflammatory disease, an immunologically-mediated disease, a viral disease, or a bone disease. The exact amount required 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 in which it is administered, 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.

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, etc., 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

As generally described above, the compounds of the present invention are useful as inhibitors of protein kinases. In one embodiment, the compounds and compositions of the present invention are inhibitors of one or more of PIM-1, CDK-2, SRC or GSK-3, and thus, without wishing to be bound by any particular theory, the compounds and compositions are particularly useful for treating or lessening the severity of a disease, condition or disorder in which activation of one or more of PIM-1, CDK-2, SRC or GSK-3 is implicated. When activation of PIM-1, CDK-2, SRC or GSK-3 is implicated in a particular disease, condition or disorder, the disease, condition or disorder may also be referred to as a "PIM-1, CDK-2, SRC or GSK-3-mediated disease" or disease symptom. Thus, in another aspect, the invention provides methods of treating or lessening the severity of a disease, disorder or condition in which activation of one or more of PIM-1, CDK-2, SRC or GSK-3 is implicated in the disease state.

The activity of a compound used as a PIM-1, CDK-2, SRC or GSK-3 inhibitor in the present invention may be determined in vitro, in vivo or in a cell line. In vitro assays include determining inhibition of phosphorylation activity or ATPase activity of activated PIM-1, CDK-2, SRC or GSK-3. A selective in vitro assay quantifies the ability of an inhibitor to bind to PIM-1, CDK-2, SRC or GSK-3. Binding of the inhibitor can be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/PIM-1, CDK-2, SRC or GSK-3 complex and determining the amount of radiolabel bound. Alternatively, binding of the inhibitor may be determined by incubating the novel inhibitor with PIM-1, CDK-2, SRC or GSK-3 bound to a known radioligand in a competition experiment.

The term "measurably inhibits" as used herein means that there is a measurable change in PIM-1, CDK-2, SRC or GSK-3 activity between a sample comprising the composition and PIM-1, CDK-2, SRC or GSK-3 kinase and an equivalent sample comprising PIM-1, CDK-2, SRC or GSK-3 without the presence of the composition.

The term "PIM-mediated disease" as used herein means any disease or other deleterious condition in which PIM family kinases are known to play a role. Such disorders include, without limitation, cancer, particularly lymphoma; inflammatory diseases including asthma, allergy and crohn's disease; and immunosuppression, including transplant rejection and autoimmune diseases.

The term "CDK-2-mediated disease" as used herein means any disease or deleterious condition in which CDK-2 is known to play a role. Accordingly, these compounds are useful in the treatment of diseases or conditions known to be affected by CDK-2 kinase activity. Such diseases or conditions include cancer, alzheimer' S disease, restenosis, angiogenesis, glomerulonephritis, cytomegalovirus, HIV, herpes, psoriasis, atherosclerosis, hair loss, and autoimmune diseases, such as rheumatoid arthritis, viral infections, neurodegenerative diseases, diseases associated with thymocyte apoptosis, or proliferative diseases resulting from cell cycle disorders, particularly progression from stage G1 to stage S.

The term "GSK-3-mediated disease" as used herein means any disease or other deleterious condition in which GSK-3 is known to play a role. Such diseases or conditions include, without limitation, autoimmune diseases, inflammatory diseases, metabolic, neurological and neurodegenerative diseases (e.g., alzheimer's Disease, huntington's Disease, parkinson's Disease, basal ganglia dyskinesia, chorea, dystonia, wilson's Disease, pick's Disease, frontal lobe degeneration, Progressive Supranuclear Palsy (PSP), creutzfeldt-Jakob Disease (Greutzfeldt-Jakob Disease), tauopathies, and corticobasal degeneration (CBD)), psychiatric disorders (e.g., schizophrenia, AIDS-related dementia, depression, bipolar disorders, and anxiety disorders), cardiovascular diseases, allergies, asthma, diabetes, amyotrophic lateral sclerosis (AML, Lou Gehrig's Disease), Multiple Sclerosis (MS), myocardial hypertrophy, reperfusion/ischemia, stroke, and alopecia.

The term "Src-mediated disease" as used herein means any disease or other deleterious condition in which Src kinase plays a role. Such diseases or conditions include, without limitation, cancers such as colon, breast, liver and pancreatic cancers; autoimmune diseases, such as transplant rejection, allergies, rheumatoid arthritis, leukemia; bone remodeling diseases, such as osteoporosis; and viral diseases such as hepatitis b infection.

In other embodiments, the invention relates to a method of enhancing glycogen synthesis and/or lowering blood glucose levels in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a composition comprising a compound of formula I. The method is particularly useful for diabetic patients.

In another embodiment, the invention relates to a method of inhibiting the production of hyperphosphorylated tau protein in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a composition comprising a compound of formula I. The method is particularly useful for stopping or delaying the progression of Alzheimer's disease.

In another embodiment, the invention relates to a method of inhibiting β -catenin phosphorylation in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a composition comprising a compound of formula I. The method is particularly useful for treating schizophrenia.

In an alternative embodiment, the method of the invention employing a composition that does not contain an additional therapeutic agent comprises the additional step of separately administering the additional therapeutic agent to the patient. When these additional therapeutic agents are administered separately, they may be administered to the patient prior to, concurrently with, or subsequent to the administration of the compositions of the present invention.

It will also be appreciated that the compounds and pharmaceutically acceptable compositions of the present invention may be used in combination therapy, that is, the compounds and pharmaceutically acceptable compositions may be administered simultaneously, prior to, or subsequent to one or more other desired therapeutic agents or pharmaceutical procedures. The particular combination of therapies (therapeutic agents or procedures) used in the combination regimen will take into account the compatibility of the desired therapeutic agent and/or procedure with the desired therapeutic effect to be achieved. It will also be appreciated that the therapies used may achieve the desired effect on the same condition (e.g., the compounds of the invention may be administered simultaneously with another drug used to treat the same condition), or they may achieve different effects (e.g., control of any side effects). As used herein, an additional therapeutic agent that is normally administered to treat or prevent a particular disease or condition is said to be "appropriate for the disease or condition being treated.

For example, chemotherapeutic agents or other antiproliferative agents may be combined with the compounds of the invention to treat proliferative diseases and cancer. Other therapies or anti-cancer agents that may be used in combination with the anti-cancer agents of the present invention include surgery, radiation therapy (some examples are gamma irradiation, neutron beam radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy and systemic radioisotopes, to name a few), endocrine therapy, biological response modifiers (interferons, interleukins and Tumor Necrosis Factor (TNF), to name a few), hyperthermia and cryotherapy, drugs that attenuate any side effects (e.g., antiemetics) and other approved chemotherapeutic drugs including, but not limited to, alkylating drugs (nitrogen mustards, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (methotrexate), purine and pyrimidine antagonists (6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine), fusioninhibitors (vinblastine, gemcitabine), and other chemotherapeutic agents, Vincristine, vinorelbine, paclitaxel), podophyllotoxin (etoposide, irinotecan, topotecan), antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas (nitrosourea mustard, cyclohexylnitrosourea), inorganic ions (cisplatin, carboplatin), enzymes (asparaginase), hormones (tamoxifen, leuprorelin acetate, flutamide, and megestrol), Gleevec^TMDoxorubicin, dexamethasone, and cyclophosphamide. For a more complete discussion of The most recent cancer therapies, see http:// www.nci.nih.gov/, FDA approved tumor drug list http:// www.fda.gov/cder/cancer/drug frame. htm and The Merck Manual, Seven Ed.1999, The entire contents of which are incorporated herein by reference.

Other examples of drugs that may also be combined with the inhibitors of the invention include, but are not limited to: therapeutic agents for Alzheimer's disease, e.g.Andtherapeutic agents for parkinson's disease, such as levodopa/carbidopa, entacapone, ropinirole, pramipexole, bromocriptine, pergolide, trihexyphenidyl, and amantadine; drugs for treating Multiple Sclerosis (MS), e.g. interferon-beta (e.g. interferon-beta)And)、and mitoxantrone; therapeutic agents for asthma, e.g. albuterol anddrugs for the treatment of schizophrenia, such as reptile, visfate, serekon and haloperidol; anti-inflammatory agents, such as corticosteroids, TNF blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immune modulating and immunosuppressive agents such as cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, interferon, corticosteroids, cyclophosphamide, azathioprine, and sulfasalazine; neurotrophic factors such as acetylcholinesterase inhibitors, MAO inhibitors, interferons, anticonvulsants, ion channel blockers, riluzole, and antiparkinson agents; drugs for the treatment of cardiovascular diseases, such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers and statins; drugs for treating liver diseases, such as corticosteroids, cholestyramine, interferons, and antiviral agents; drugs for treating hematological disorders, such as corticosteroids, antileukemic agents and growth factors; and drugs for the treatment of immunodeficiency disorders, such as gamma globulin.

The amount of additional therapeutic agent in the compositions of the present invention will not exceed the amount normally administered in compositions containing the therapeutic agent as the only active ingredient. Preferably, the amount of additional therapeutic agent in the presently disclosed compositions will be from about 50% to 100% of the content in typical compositions containing the drug as the sole therapeutically active ingredient.

The compounds of the present invention or pharmaceutically acceptable compositions thereof may also be incorporated into compositions for coating implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents and catheters. Thus, the present invention, in another aspect, includes a composition for coating an implantable device comprising a compound of the present invention as generally described above and as described in classes and subclasses herein, and a carrier suitable for coating said implantable device. In another aspect, the present invention includes an implantable device coated with a composition comprising a compound of the present invention as generally described above and in classes and subclasses herein, and a carrier suitable for coating the implantable device.

Vascular stents, for example, have been used to overcome restenosis (restenosis of the vessel wall after injury). However, patients using stents or other implantable devices are at risk for clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. Suitable coatings and general methods of making coated implantable devices are described in U.S. Pat. nos. 6,099,562, 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate copolymers and mixtures thereof. The coating may optionally be further covered with a surface layer of a suitable fluorosilicone, polysaccharide, polyethylene glycol, phospholipid, or combinations thereof to impart controlled release characteristics to the composition.

Another aspect of the invention relates to inhibiting PIM-1, CDK-2, SRC or GSK-3 activity in a biological sample or a patient, comprising administering to the patient or contacting said biological sample with a compound of formula I or a composition comprising said compound. The term "biological sample" as used herein includes, without limitation, cell cultures and extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of PIM-1, CDK-2, SRC or GSK-3 kinase activity in a biological sample may 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, biological sample storage, and biological assays.

In order that the invention described herein may be more fully understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any way.

Examples

Example 1: synthesis of exemplary Compounds of the invention

Some exemplary compounds, shown in scheme 2, were prepared according to the following general procedure:

the starting material (A) was dissolved in a 0.5M MeCN solution and stirred at room temperature for 2 hours. The formation of (I) was confirmed by HPLC and mass spectrometry. To the same solution was added phosphorus oxychloride in an amount equal to the volume of acetonitrile present. The reaction was allowed to proceed overnight at 80 ℃. After completion of the reaction, the mixture was poured onto ice and stirred for 1 hour. After water treatment and extraction with ethyl acetate, the organic layer was dried over magnesium sulfate, filtered, and concentrated to dryness. The crude product was purified by silica gel column chromatography.

40-400mg of (II) are dissolved in 0.5 to 1.0mL of a cycloalkylamine and heated to 70 ℃ for 4 hours. The reaction was concentrated to dryness and purified by reverse phase preparative HPLC.

Certain exemplary compounds, as shown in scheme 3, were prepared according to the following general procedure:

(II) was dissolved in 1M THF solution, followed by the addition of 5 equivalents glycine methyl ester hydrochloride and 5 equivalents triethylamine. The reaction tube was sealed and heated to 130 ℃ for 48 hours. The reaction was concentrated to dryness, extracted with ethyl acetate and water, over MgSO₄Drying and concentrating the solution to dryness. The product was purified by reverse phase preparative HPLC.

(IIIA) was dissolved in 1M aqueous NaOH and heated to reflux for 2 hours. The crude reaction was concentrated to dryness. The product (IV) was purified by reverse phase preparative HPLC.

Dissolving (IIIA) in 7N NH₃Heated to reflux for 4 hours. The crude reaction was concentrated to dryness. The product (V) was purified by reverse phase preparative HPLC.

Certain exemplary compounds shown in scheme 4 were prepared according to the following general procedure:

starting materials A and I are commercially available, and preparation of intermediate IIA is described in "A MildAbach to 1, 3, 4-oxadiazines and Fused 1, 2, 4-Triazoles. diazenes as Intermediates? "Janez Kosmrlj, Marijan Kocevar, Slovenko Polanc, Synlett, 7, 652 (1996).

General experiments:

(VII): starting material (IIA) was dissolved in 1M N-methylpyrrolidone solution, followed by addition of (VI) and heating to 100 ℃ overnight. The reaction was complete by HPLC and MS + check, then concentrated to dryness. The product was purified by preparative reverse phase HPLC.

(VIII): the starting material (IIA) was dissolved in methanol containing 5 equivalents of sodium methoxide. The reaction was heated to reflux for 4 hours. The crude product was purified by preparative reverse phase HPLC.

(IX): starting material (IIA) was dissolved in 500. mu.l cyclopropylamine. The tube was sealed and heated to 70 ℃ for 4 hours. Once the reaction was complete, the reaction was concentrated to dryness and then purified by preparative reverse phase HPLC.

Table 2 below depicts exemplary data for certain compounds of the present invention:

compound numbering	MSpos	NMR
compound numbering	MSpos	NMR	I-5	320.2	DMSO-d6：8.80(d，2H)，8.07(d，1H)，7.98(d，2H)，7.90(s，1H)，6.85(d，1H)，2.75(m，1H)，0.90(m，2H)，0.58(m，2H)
I-14	269.2	DMSO-d6：8.52(m，2H)，8.04(d，1H)，7.90(m，2H)，7.65(s，1H)，7.35(m，2H)，7.20(s，1H)，7.00(d，1H)，3.89(d，2H)	I-5	320.2
I-14	269.2		I-15	252.2	MeOH-d4：8.55(m，2H)，7.98(d，1H)，7.60(m，3H)，7.07(d，1H)，2.78(m，1H)，0.92(m，2H)，0.65(m，2H)
I-16	270.1	MeOH-d4：8.45(d，2H)，7.85(d，1H)，7.57(t，2H)，7.50(t，1H)，7.00(d，1H)，3.95(s，2H)	I-15	252.2
I-16	270.1		I-17	320.1	MeOH-d4：7.97(m，1H)，7.93(d，1H)，7.84(m，2H)，7.77(m，1H)，7.00(d，1H)，2.50(m，1H)，0.65(m，2H)，0.46(m，2H)
I-18	320.2	DMSO-d6：9.15(s，1H)，8.75(d，1H)，8.07(d，1H)，7.92(s，1H)，7.88(d，1H)，7.81(t，1H)，6.84(d，1H)，2.70(m，1H)，0.84(m，2H)，0.59(m，2H)	I-17	320.1
I-18	320.2		I-19	270.1	DMSO-d6：8.05(m，2H)，7.75(s，1H)，7.62(m，1H)，7.45(m，2H)，6.86(d，1H)，2.59(m，1H)，0.72(m，2H)，0.50(m，2H)
I-20	270.1	DMSO-d6：8.48(d，1H)，8.40(d，1H)，8.05(d，1H)，7.87(s，1H)，7.62(m，1H)，7.36(m，1H)，6.82(d，1H)，2.70(m，1H)，0.85(m，2H)，0.60(m，2H)	I-19	270.1
I-20	270.1		I-21	270.1	DMSO-d6：8.62(m，2H)，8.00(d，1H)，7.81(s，1H)，7.42(m，2H)，6.80(d，1H)，2.69(m，1H)，.085(m，2H)，0.57(m，2H)

I-22	338.2	MeOH-d4：7.86(m，1H)，7.82(m，1H)，7.81(m，1H)，7.80-7.79(m，1H)，7.67(m，1H)，7.01(d，1H)，3.70(m，2H)
I-22	338.2		I-23	287.2	DMSO-d6：8.52(m，2H)，8.04(d，1H)，7.90(m，1H)，7.65(s，1H)，7.35(m，2H)，7.20(s，1H)，7.00(d，1H)，3.89(d，2H)
I-24	282.5	MeOH-d4：8.50(d，2H)，7.95(d，1H)，7.15(d，2H)，7.05(d，1H)，3.90(s，3H)，2.78(m，1H)，0.91(m，2H)，0.64(m，2H)	I-23	287.2
I-24	282.5		I-25	334.2	MeOH-d4：9.04(s，1H)，8.65(d，1H)，7.92(d，1H)，7.85(d，1H)，7.79(t，1H)，4.34(m，1H)，2.52(m，2H)，2.05(m，2H)，1.90(m，2H)
I-26	362.4	DMSO-d6：8.97(s，1H)，8.64(d，1H)，8.03(d，1H)，7.89(d，1H)，7.82(t，1H)，7.52(d，1H)，6.90(d，1H)，3.66(m，1H)，2.07(m，2H)，1.76(m，2H)，1.65(m，1H)，1.30(m，5H)	I-25	334.2
I-26	362.4		I-27	277.2	DMSO-d6：8.79(d，2H)，8.06(d，3H)，7.91(s，1H)，6.85(d，1H)，2.70(m，1H)，0.88(m，2H)，0.55(m，2H)
I-28	282.3	DMSO-d6：8.21(s，1H)，8.15(d，1H)，8.04(d，1H)，7.85(s，1H)，7.50(t，1H)，7.10(d，1H)，6.85(d，1H)，3.85(s，3H)，2.71(m，1H)，0.81(m，2H)，0.56(m，2H)	I-27	277.2
I-28	282.3		I-29	282.2	MeOH-d4：8.32(d，1H)，7.98(d，1H)，7.67(t，1H)，7.32(d，1H)，7.25(t，1H)，7.08(d，1H)，2.70(m，1H)，0.82(m，2H)，0.60(m，2H)
I-30	337.2	DMSO-d6：8.05(d，1H)，7.96(d，1H)，7.81(m，3H)，7.61(m，1H)，7.43(m，1H)，7.10(d，2H)，3.68(m，2H)	I-29	282.2
I-30	337.2		I-31	337.2	DMSO-d6：8.83(d，1H)，8.71(d，1H)，8.10(d，1H)，7.90(m，2H)，7.81(t，1H)，7.58(s，1H)，7.18(s，1H)，7.05(d，1H)，3.91(d，2H)
I-32	337.2	DMSO-d6：8.70(d，2H)，8.10(d，1H)，7.97(t，1H)，7.90(d，2H)，7.69(s，1H)，7.22(s，1H)，7.05(d，1H)，3.90(d，2H)	I-31	337.2

I-33	287.2	DMSO-d6：8.02(d，1H)，7.96(t，1H)，7.67(t，1H)，7.60(m，1H)，7.50(s，1H)，7.40(m，2H)，7.15(s，1H)，7.05(d，1H)，3.77(d，2H)
I-33	287.2		I-34	287.2	DMSO-d6：8.35(d，1H)，8.21(d，1H)，8.05(d，1H)，7.89(t，1H)，7.61(m，2H)，7.35(t，1H)，7.20(s，1H)，7.05(d，1H)，3.90(d，2H)
I-35	338.1	DMSO-d6：8.78(s，1H)，8.70(d，1H)，8.08(m，2H)，7.89(d，1H)，7.80(m，1H)，7.05(d，1H)，4.05(d，2H)	I-34	287.2
I-35	338.1		I-36	277.2	DMSO-d6：9.13(s，1H)，8.78(d，1H)，8.05(d，1H)，7.95(m，2H)，7.81(t，1H)，6.85(d，1H)，2.70(m，1H)，0.85(m，2H)，0.50(m，2H)
I-37	294.2	DMSO-d6：8.65(d，2H)，8.10(d，1H)，7.95(m，3H)，7.67(s，1H)，7.20(s，1H)，7.05(d，1H)，3.90(d，2H)	I-36	277.2
I-37	294.2		I-38	299.2	DMSO-d6：8.40(d，2H)，8.00(d，1H)，7.78(t，1H)，7.62(s，1H)，7.20(s，1H)，7.09(d，2H)，6.96(d，1H)，3.87(d，2H)，3.84(s，3H)
I-39	299.2	DMSO-d6：8.08(d，1H)，8.03(d，1H)，8.01(m，1H)，7.80(t，1H)，7.59(s，1H)，7.46(t，1H)，7.18(s，1H)，7.07(d，1H)，7.01(d，1H)，3.91(d，2H)，3.87(s，3H)	I-38	299.2
I-39	299.2		I-40	294.2	DMSO-d6：8.80(d，1H)，8.72(s，1H)，8.08(d，1H)，7.98(d，1H)，7.95(t，1H)，7.76m，1H)，7.61s，1H)，7.15(s，1H)，7.05(d，1H)，3.92(d，2H)
I-42	242	DMSO-d6：7.60(d，1H)，7.49(d，2H)，6.95(t，2H)，6.35(d，1H)，6.30(t，1H)，3.93(s，3H)	I-40	294.2
I-42	242		I-43	319.97	DMSO-d6：9.50(s，1H)，8.05(d，1H)，7.75(d，2H)，7.65(m，2H)，7.55(m，3H)，7.35(t，2H)，6.95(t，1H)，6.69(d，1H)
I-44	304.02	DMSO-d6：9.17(s，1H)，8.26(d，1H)，7.66(d，2H)，7.46(t，2H)，7.33(d，2H)，7.28(m，3H)，7.02(d，1H)，6.92(t，1H)	I-43	319.97
I-44	304.02		I-45	260.01	MeOH-d4：7.90(d，1H)，7.68(m，2H)，7.07(m，2H)，6.89(d，1H)，4.1(s，3H)

I-46	260	MeOH-d4：7.90(d，1H)，7.58(d，1H)，7.45(d，1H)，7.30(m，1H)，6.90(d，1H)，6.70(t，1H)，4.1(s，3H)
I-46	260		I-47	260.01	DMSO-d6：8.50(s，1H)，8.13(d，1H)，7.85(t，1H)，7.25(m，1H)，7.16(t，1H)，7.01(m，1H)，6.93(d，1H)，3.97(s，3H)
I-48	337.97	DMSO-d6：9.60(s，1H)，8.05(d，1H)，7.85(m，2H)，7.67(m，2H)，7.52(m，3H)，7.18(t，2H)，6.68(d，1H)	I-47	260.01
I-48	337.97		I-49	337.95	DMSO-d6：9.83(s，1H)，8.06(d，1H)，7.73(d，1H)，7.67(m，2H)，7.60(d，1H)，7.51(m，3H)，7.34(m，1H)，6.75(t，1H)，6.70(d，1H)
I-50	337.97	DMSO-d6：8.44(s，1H)，8.10(d，1H)，7.80(t，1H)，7.65(d，2H)，7.50(m，3H)，7.27(t，1H)，7.15(t，1H)，7.03(m，1H)，6.94(d，1H)	I-49	337.95
I-50	337.97		I-51	309.99	DMSO-d6：9.59(s，1H)，8.15(d，1H)，7.95(d，2H)，7.70(d，2H)，6.95(d，1H)，4.05(s，3H)
I-52	309.99	DMSO-d6：9.50(s，1H)，8.13(s，1H)，8.15(d，1H)，8.08(d，1H)，7.58(t，1H)，7.29(d，1H)，6.95(d，1H)，4.10(s，3H)	I-51	309.99
I-52	309.99		I-53	309.98	DMSO-d6：8.19(d，1H)，8.02(s，1H)，7.91(d，1H)，7.71(d，1H)，7.65(t，1H)，7.21(t，1H)，7.00(d，1H)，3.90(s，3H)
I-54	322.02	DMSO-d6：9.30(s，1H)，8.27(d，1H)，7.69(m，2H)，7.45(m，2H)，7.33(d，2H)，7.27(t，1H)，7.15(t，2H)，7.05(d，1H)	I-53	309.98
I-54	322.02		I-55	322.01	DMSO-d6：9.55(s，1H)，8.29(d，1H)，7.59(d，1H)，7.451(m，3H)，7.35-7.25(m，4H)，7.05(d，1H)，6.72(t，1H)
I-56	322.04	DMSO-d6：8.51(s，1H)，8.31(d，1H)，7.59(t，1H)，7.41(t，2H)，7.33-7.11(m，6H)，7.01(m，1H)	I-55	322.01
I-56	322.04		I-57	388.05	DMSO-d6：10.07(s，1H)，8.10(d，1H)，7.92(d，2H)，7.67(m，4H)，7.50(m，3H)，6.75(d，1H)

I-58	388.04	DMSO-d6：10.5(s，1H)，8.3(s，1H)，8.1(d，1H)，8.05(d，1H)，7.67(m，2H)，7.58-7.49(m，4H)，7.29(d，1H)，6.71(d，1H)
I-58	388.04		I-59	388.05	DMSO-d6：8.17(d，1H)，8.00(d，1H)，7.82(s，1H)，7.68(d，1H)，7.63(m，3H)，7.53(m，1H)，7.47(t，2H)，7.19(t，1H)，7.14(d，1H)
I-60	350	DMSO-d6：9.26(s，1H)，8.02(d，1H)，7.75(d，2H)，7.66(m，2H)，7.53(m，3H)，6.91(d，2H)，3.75(s，3H)	I-59	388.05
I-60	350		I-63	334.02	DMSO-d6：9.05(s，1H)，8.25(d，1H)，7.60(d，2H)，7.50(m，2H)，7.35(m，3H)，7.05(d，1H)，6.95(m，2H)，3.79(s，3H)
I-61	349.99	DMSO-d6：9.55(s，1H)，8.05(d，1H)，7.70(m，2H)，7.51(m，3H)，7.45(s，1H)，7.35(d，1H)，7.24(t，1H)，6.73(d，1H)，6.57(d，1H)，3.75(s，3H)	I-63	334.02
I-61	349.99		I-64	334.03	DMSO-d6：9.20(s，1H)，8.25(d，1H)，7.48(m，2H)，7.35-7.18(m，6H)，7.05(d，1H)，6.52(d，1H)，3.75(s，3H)
I-62	350	DMSO-d6：8.20(m，1H)，8.15(d，1H)，7.75(d，2H)，7.68-7.58(m，4H)，7.15(d，1H)，7.05(m，1H)，7.00(m，2H)，3.85(s，3H)	I-64	334.03
I-62	350		I-66	272.03	DMSO-d6：8.80(s，1H)，8.05(d，1H)，7.73(d，2H)，6.92(d，2H)，6.87(d，1H)，4.05(s，3H)，3.75(s，3H)
I-67	272.04	DMSO-d6：9.00(s，1h)，8.10(d，1H)，7.47(s，1H)，7.37(d，1H)，7.22(t，1H)，6.90(d，1H)，6.55(d，1H)，4.05(s，3H)，3.86(s，3H)	I-66	272.03
I-67	272.04		I-68	272.04	DMSO-d6：8.21(d，1H)，7.65(s，1H)，7.10(d，1H)，7.05-6.95(m，3H)，4.05(s，3H)，3.95(s，3H)
I-69	344.9	DMSO-d6：10.2(s，1H)，8.12(d，1H)，7.90(d，2H)，7.75(d，2H)，7.65(d，2H)，7.55(m，3H)，6.75(d，1H)	I-68	272.04
I-69	344.9		I-70	328.04	DMSO-d6：9.65(d，2H)，8.01(d，1H)，7.75(m，4H)，7.65(d，2H)，7.19(t，2H)，7.00(m，2H)

I-71	344.9	DMSO-d6：10.05(s，1H)，8.25(s，1H)，8.11(d，1H)，8.06(d，1H)，7.67(d，2H)，7.50(m，4H)，7.40(m，1H)，6.75(d，1H)
I-71	344.9		I-72	329	DMSO-d6：9.71(s，1H)，8.28(d，1H)，8.05(s，1H)，7.9(d，1H)，7.45-7.22(m，7H)，7.09(d，1H)
I-73	267.04	DMSO-d6：9.70(s，1H)，8.15(d，1H)，7.87(d，2H)，7.75(d，2H)，6.96(d，1H)，4.05(s，3H)	I-72	329
I-73	267.04		I-74	267.04	DMSO-d6：9.52(s，1H)，8.25(s，1H)，8.15(d，1H)，8.05(d，1H)，7.55(t，1H)，7.39(d，1H)，6.96(d，1H)，4.05(s，3H)
I-75	292	DMSO-d6：9.45(s，1H)，7.84(d，1H)，7.80-7.70(m，4H)，7.65(d，1H)，6.72(d，1H)，2.80(m，1H)，0.70(m，2H)，0.50(m，2H)	I-74	267.04
I-75	292		I-76	292	DMSO-d6：9.19(s，1H)，8.12(s，1H)，7.99(d，1H)，7.82(d，1H)，7.53(m，2H)，7.35(m，2H)，6.68(d，1H)，2.82(m，1H)，0.79(m，2H)，0.51(m，2H)
I-77	285	MeOH-d4：7.89(d，1H)，7.52(d，1H)，7.45(d，1H)，7.38(m，1H)，7.18(d，1H)，6.83(t，1H)，2.94(m，1H)，0.90(m，2H)，0.62(m，2H)	I-76	292
I-77	285		I-78	285	MeOH-d4：7.81(d，1H)，7.62(m，2H)，7.15(m，3H)，2.90(m，1H)，0.88(m，2H)，0.60(m，2H)
I-79	285	MeOH-d4：7.81(m，2H)，7.26(m，3H)，7.05(d，1H)，2.82(m，1H)，0.85(m，2H)，0.60(m，2H)	I-78	285
I-79	285		I-80	335	MeOH-d4：7.85(m，3H)，7.66(d，2H)，7.12(d，1H)，2.90(m，1H)，0.90(m，2H)，0.60(m，2H)
I-81	335	MeOH-d4：8.07(s，1H)，7.90(m，2H)，7.56(t，1H)，7.40(d，1H)，7.18(d，1H)，2.93(m，1H)，0.90(m，2H)，0.61(m，2H)	I-80	335
I-81	335		I-82	297	MeOH-d4：7.83(d，1H)，7.33-7.10(m，4H)，6.74(d，1H)，3.81(s，3H)，2.90(m，1H)，0.90(m，2H)，0.64(m，2H)

I-83	297	MeOH-d4：7.86(d，1H)，7.80(d，1H)，7.19(m，1H)，7.15(d，1H)，7.05(m，2H)，3.95(s，3H)，2.80(m，1H)，0.89(m，2H)，0.65(m，2H)
I-83	297		I-84	302	DMSO-d6：8.15(d，1H)，7.95(s，1H)，7.65(s，1H)，7.02(d，1H)，6.95(d，1H)，6.54(d，1H)，4.05(s，3H)，3.90(s，3H)，3.80(s，3H)
I-85	302	DMSO-d6：8.40(s，1H)，8.16(d，1H)，7.81(d，1H)，7.05(d，1H)，6.75(s，1H)，6.60(d，1H)，4.05(s，3H)，3.90(s，3H)，3.80(s，3H)	I-84	302
I-85	302		I-86	332	DMSO-d6：8.90(s，1H)，8.10(d，1H)，7.30(s，2H)，6.90(d，1H)，4.10(s，3H)，3.83(s，6H)，3.67(s，3H)

Example 2: inhibition of PIM-1

Compounds were screened for their ability to inhibit PIM-1 using a standard conjugated enzyme assay (Fox et al (1998) Protein Sci 7, 2249). The reaction was at 100mM HEPES pH 7.5, 10mM MgCl₂25mM NaCl, 1mM DTT, 20. mu.g/ml BSA and 1.5% DMSO. The final substrate concentrations in the assay were 120. mu.M ATP (Sigma Chemicals) and 200. mu.M Peptide (American Peptide, Sunnyvale, Calif.). The measurement was carried out at 30 ℃ and 50 nMIPM-1. The final concentration of the components of the coupled enzyme system was 2.5mM phosphoenolpyruvate, 350. mu.M NADH, 30. mu.g/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

Assay stock buffer solutions were prepared containing all the above reagents except PIM-1, DTT, BSA and related test compounds. Mu.l of the test reaction was placed in 384 well plates followed by the addition of 1. mu.l of 2mM DMSO stock solution containing the test compound (final compound concentration 30. mu.M). The plate was preincubated at 30 ℃ for about 10 minutes, and 10. mu.l of DTT containing enzyme and BSA were added to initiate the reaction (final concentration: 50nM PIM-1, 1mM DTT and 20. mu.g/ml BSA). Reaction rates were obtained using a BioRad Ultramark plate reader (Hercules, CA) at 30 ℃ over a 5 minute reading time. Titration of compounds showing > 50% inhibition relative to standard wells containing DMSO without compound, IC was determined using a similar protocol₅₀。

The compounds of the present invention were shown to inhibit PIM-1 using the assay methods described above. In certain embodiments, the following compounds exhibit an IC for PIM-1 of less than 1.0. mu.M₅₀Or K_iThe value: i-5, I-18, I-20, I-21, I-24, I-25, I-26 and I-28.

Example 3: inhibition of GSK-3

Inhibition of GSK-3 β (AA 1-42) against it using a standard conjugated enzyme assay (Fox et al (1998) Protein Sci 7, 2249)0) Ability to screen compounds. The reaction was carried out in solution with 100mM HEPES pH 7.5, 10mM MgCl₂25mM NaCl, 300. mu.M NADH, 1mM DTT and 1.5% DMSO. The final substrate concentrations in the assay were 20. mu.M ATP (Sigma Chemicals, St Louis, Mo.) and 300. mu.M Peptide (American Peptide, Sunnyvale, Calif.). The assay was performed at 30 ℃ and 20nM GSK-3 β. The final concentration of the components of the coupled enzyme system was 2.5mM phosphoenolpyruvate, 300. mu.M NADH, 30. mu.g/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

Assay stock buffer solutions were prepared containing all the above reagents, with the exception of ATP and related test compounds. Assay stock buffer (175. mu.l) was incubated in 96-well plates for 10 minutes at 30 ℃ with 5. mu.l of test compound at a final concentration varying from 0.002. mu.M to 30. mu.M. Typically, serial dilutions (starting from a 10mM stock solution of the compound) are prepared in daughter plates with DMSO of the test compound and a 12-point titration is performed. The reaction was initiated by adding 20. mu.l of ATP (final concentration: 20. mu.M). The reaction rate was obtained over 10 min at 30 ℃ using a Molecular devices Spectramax plate reader (Sunnyvale, Calif.). Determination of K from Rate data as a function of inhibitor concentration_iThe value is obtained.

The compounds of the invention were shown to inhibit GSK-3 using the assay described above. In certain embodiments, the following compounds exhibit a K of less than 2.0 μ M for GSK-3_iThe value: i-18, I-21, I-37, I-38, I-39, I-40, I-62, I-66, I-85 and I-86.

Example 4: CDK-2 inhibition

Compounds were screened for their ability to inhibit CDK-2/cyclin A using a standard coupled enzyme assay (Fox et al (1998) Protein Sci 7, 2249). The reaction was carried out at 100mM HEPES pH 7.5, 10mM MgCl₂25mM NaCl, 1mM DTT and 1.5% DMSO. The final substrate concentrations in the assay were 100. mu.M ATP (Sigma Chemicals) and 100. mu.M Peptide (American Peptide, Sunnyvale, Calif.). The assay was performed at 30 ℃ and 25 nCDK-2/cyclin A. The final concentration of the components of the coupled enzyme system was 2.5mMPhosphoenolpyruvate, 350. mu.M NADH, 30. mu.g/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

Assay stock buffers were prepared containing all of the above reagents, except CDK-2/cyclin A, DTT and the relevant test compounds. Mu.l of the test reaction was placed in 384-well plates followed by the addition of 1. mu.l of 2mM DMSO stock solution containing the test compound (final compound concentration 30. mu.M). The plate was preincubated at 30 ℃ for about 10 minutes, and 10. mu.l of enzyme was added to initiate the reaction (final concentration: 25 nM). Reaction rates were obtained using a BioRad Ultramark plate reader (Hercules, CA) at 30 ℃ over a 5 minute reading time. Determination of K according to Standard methods_iThe value is obtained.

The compounds of the invention were shown to inhibit CDK-2 using the assay described above. In certain embodiments, the following compounds exhibit a K of less than 2.0 μ M for CDK-2_iThe value: i-68 and I-73.

Example 5: suppression of SRC

The compounds are evaluated for human Src kinase inhibitors using a radioactivity-based assay or a spectrophotometric assay.

Src inhibition assay a: radioactivity-based assays

The compounds tested were inhibitors of full-length recombinant human Src kinase (from Upstate Biotechnology, cat. No.14-117) expressed and purified in baculovirus cells. In that³³Src kinase activity is monitored after P incorporates from ATP tyrosine, with a composition of Glu: Tyr ═ 4: 1, random polyglu-Tyr polymer substrate (Sigma, cat. No. P-0275). The final concentrations of the assay components are as follows: 0.05M HEPES, pH 7.6, 10mM MgCl₂，2mM DTT，0.25mg/mlBSA，10μM ATP(1-2μCi ³³P-ATP per reaction), 5mg/ml of poly Glu-Tyr and 1-2 units of recombinant human Src kinase. In a typical assay, all reaction components except ATP are premixed and aliquoted into assay plate wells. A DMSO solution of inhibitor was added to the wells to give a final DMSO concentration of 2.5%. The assay plate was incubated at 30 ℃ for 10 minutes,then use³³P-ATP initiates the reaction. After 20 minutes of reaction, the reaction mixture was washed with a solution containing 20mM Na₃PO₄The reaction was quenched with 150. mu.l of 10% trichloroacetic acid (TCA). The quenched sample was then transferred to a 96-well Filter plate (Wha tman, UNI-Filter GF/F Glass Fiber Filter, cat No.7700-3310) mounted on a Filter plate vacuum manifold. The filter plate was washed with 20mM Na₃PO₄The 10% TCA was washed four times, then four times with methanol. Then 200 μ l scintillation fluid was added to each well. The plate was sealed and the amount of radioactivity associated with the filter was quantified on a TopCount scintillation counter. Incorporated radioactivity was plotted against inhibitor concentration. Bringing the data into a competitive inhibition kinetics model to obtain K of the compound_i。

Src inhibition assay B: spectrophotometry

ADP produced from ATP by phosphorylation of the human recombinant Src kinase-catalyzed polyglulu-Tyr substrate was quantified using a coupled enzyme assay (Fox et al (1998) Protein Sci 7, 2249). In the present assay, for each molecule of ADP generated in the kinase reaction, one molecule of NADH is oxidized to NAD. The disappearance of NADH is suitably observed at 340 nm.

The final concentrations of the assay components are as follows: 0.025M HEPES, pH 7.6, 10mM MgCl2, 2mM DTT, 0.25mg/ml polyGlu-Tyr and 25nM recombinant human Src kinase. The final concentration of the components of the coupled enzyme system was 2.5mM phosphoenolpyruvate, 200. mu.M NADH, 30. mu.g/ml pyruvate kinase and 10. mu.g/ml lactate dehydrogenase.

In a typical assay, all reaction components except ATP are premixed and aliquoted into assay plate wells. A DMSO solution of inhibitor was added to the wells to give a final DMSO concentration of 2.5%. The assay plates were incubated at 30 ℃ for 10 minutes and then the reaction was initiated with 100 μ MATP. The change in absorbance at 340nm over time, i.e., the rate of reaction, was monitored on a molecular device plate reader. Substituting rate data as a function of inhibitor concentration into a competitive inhibition kinetics model to obtain K for the compound_i。

The compounds of the present invention were shown to inhibit SRC using the assay methods described above. In certain embodiments, the following compounds exhibit a K of less than 5.0 μ M for SRC_iThe value: i-25, I-26, I-28 and I-29.

Claims

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof,

wherein R is¹Is NR³R⁴(ii) a Wherein R is⁴Is hydrogen, R³Is phenyl or C_3-7A cycloalkyl group;

R²is- (T)_nAr¹Wherein T is NR, wherein R is hydrogen; n is 0 or 1; and Ar¹Is optionally- (QR)^x)_xSubstituted phenyl;

wherein x is 0, 1 or 2 and Q is a bond; each occurrence of R^XIndependently selected from halogen, R ', CN OR' optionally substituted with halogen;

wherein R' is C_1-8An alkyl group;

the conditions are as follows:

when R is¹When is-NH (cyclopropyl), then R²CF not appearing once in para position₃A substituted phenyl group.

2. The compound of claim 1, wherein R⁴Is hydrogen; r²is-NRAr¹Wherein R is hydrogen, said compound having one of the following formulae:

wherein y is 0.

3. The compound of claim 1, wherein R²Is NRAr¹Wherein R is hydrogen, Ar¹Is an optionally substituted phenyl group, said compound having the formula:

wherein x is 0, 1 or 2.

4. The compound of claim 1, having one of the following structures:

5. a pharmaceutical composition for inhibiting PIM-1, GSK-3, CDK-2, or SRC kinase activity comprising a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or excipient.

6. Use of a compound according to any one of claims 1 to 4 in the preparation of an agent for inhibiting PIM-1, GSK-3, CDK-2 or SRC kinase activity in a biological sample.

7. The use of claim 6, wherein the agent is for inhibiting PIM-1 activity.

8. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or lessening the severity of a disease or condition selected from chronic myelogenous leukemia, acute promyelocytic leukemia, rheumatoid arthritis, asthma, osteoarthritis, ischemia, cancer, liver disease, myocardial infarction, congestive heart failure, pathological immune conditions involving T cell activation, and neurodegenerative diseases.