HK1260037B - Substituted tricyclic herteocyclic compounds and use thereof - Google Patents

Description

Substituted tricyclic heterocyclic compounds and uses thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefits of international patent application No. PCT/CN2016/085811 filed with the State Intellectual Property Office on June 15, 2016, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of pharmaceutical technology, and more specifically relates to compounds, compositions comprising the same, and their use for treating conditions caused by cancer and neurodegenerative diseases. In particular, provided herein are substituted tricyclic heterocyclic compounds capable of inhibiting more than one kinase, particularly LRRK, more particularly LRRK2.

Background Art

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting 1-2% of the elderly population [1]. Genome-wide association studies (GWAS) have identified 28 genetic risk variants at 24 loci associated with non-familial PD [2]. Among them, mutations in LRRK2 (Park8) have been found to be inherited, identifying shared molecular pathways that drive pathogenesis in familial and non-familial PD and include the most common cause of the disease [3,4]. The PD-causing LRRK2 mutation profile is primarily associated with the kinase (G2019S, I2020T) and ROC-COR domains (R1441C/G/H, Y1699C), indicating that these enzymatic activities are critical for pathogenesis [5]. The frequency of pathogenic mutations is rare, at approximately 2% overall [6,7], however, the most common mutation, G2019S, has been found in up to 40% of patients in certain ethnic populations, activating the kinase by 2- to 3-fold [8-13]. In addition to pathogenic mutations, common genetic variability in LRRK2 is a risk factor for sporadic PD [14-16].

In 2004, LRRK2 was identified as the gene responsible for the inheritance of PD associated with the PARK8 locus [17,18] and was found to consist of 51 exons, producing a large (268 kDa) protein. Subsequently, many variants in the primary structure of LRRK2 were identified, including dominant mutations that segregate with familial PD and also occur in sporadic PD, as well as polymorphisms at the LRRK2 locus that increase the lifetime risk of developing sporadic PD [20-22].

LRRK2 is a multidomain protein that contains two enzymatic functions at its core. A GTPase domain consisting of a Ras complex protein (ROC) terminated by a spacer domain at the C-terminus (COR) called the Roc domain is followed by a kinase domain belonging to a serine/threonine kinase. This enzymatic core is surrounded by a protein-protein interaction domain containing armadillo, ankyrin, and leucine-rich repeat (LRR) domains at the N-terminus of LRRK2 [23]. The C-terminus of LRRK2 contains a WD40 domain, which is thought to be essential for protein folding, thereby controlling LRRK2 function and kinase activity [24]. Interestingly, the major pathogenic mutations described to date occur within the enzymatic core of LRRK2, suggesting that modifications in LRRK2 activity significantly influence the onset and progression of PD.

To date, nearly 40 single amino acid substitution mutations have been associated with autosomal dominant PD [25,26]. The most common LRRK2 mutation, which accounts for approximately 6% of familial PD in Europe and 3% of sporadic PD cases, involves an amino acid substitution of Gly2019 to Ser residue. Gly2019 is located within the conserved DYG-Mg2 ⁺ -binding motif in subdomain-VII of the kinase domain [25]. Recent reports indicate that this mutation enhances LRRK2 autophosphorylation and its ability to phosphorylate myelin basic protein 2-3 fold [8,12,27]. When G2019S-LRRK2 is overexpressed in cell lines and primary neuronal cultures, cytotoxicity in the absence and presence of oxidative stress, as well as the formation of inclusion bodies, was observed [27,28]. These results, together with the fact that genetic inactivation of LRRK2 kinase activity showed a protective effect against this toxic phenotype, suggest that alterations in LRRK2 kinase activity are potentially involved in the neurotoxic and pathogenic mechanisms of LRRK2-PD.

Induced pluripotent stem cells (iPSCs) derived from patients with LRRK2G2019S Parkinson's disease have been found to exhibit defects in neurite outgrowth and increased sensitivity to rotenone, which can be ameliorated by genetic correction of the G2019S mutation or treatment of cells with small molecule inhibitors of LRRK2 kinase activity [29]. Increased mitochondrial damage associated with the LRRK2G2019S mutation in iPSCs was also blocked by genetic correction of the G2019S mutation [30].

Additional evidence links LRRK2 function and dysfunction to the autophagy-lysosomal pathway [31]. LRRK2 protein leads to defects in chaperone-mediated autophagy, which negatively impacts the ability of cells to degrade α-synuclein [32]. In other cell models, selective LRRK2 inhibitors have been shown to stimulate macrophage phagocytosis [33]. These data suggest that small molecule inhibitors of LRRK2 kinase activity could be effective in treating diseases characterized by cellular proteolytic defects caused by aberrant autophagy/lysosomal degradation pathways, including Parkinson's disease associated with GBA mutations [34], other α-synucleinopathies, tauopathies, Alzheimer's disease [35] and other neurodegenerative diseases [36], and Gaucher disease [37]. Furthermore, significantly elevated levels of LRRK2 mRNA have been observed in fibroblasts from patients with Niemann-Pick disease type C (NPC) compared with fibroblasts from normal subjects, suggesting that aberrant LRRK2 function may play a role in lysosomal diseases [38]. This observation suggests that LRRK2 inhibitors may be effective in treating NPC.

The G2019S mutant form of LRRK2, which is associated with PD, has also been reported to enhance the phosphorylation of tubulin-associated Tau [39], and a disease model in which LRRK2 acts upstream of the pathogenic effects of Tau and α-synuclein has been proposed [40]. In support of this, LRRK2 expression in a transgenic mouse model is associated with increased insoluble Tau aggregation and increased Tau phosphorylation [41]. Overexpression of the PD pathogenic mutant protein LRRK2R1441G has been reported to result in parkinsonian symptoms and Tau hyperphosphorylation in a transgenic mouse model [42]. Therefore, these data suggest that LRRK2 inhibitors with kinase catalytic activity may be useful for the treatment of tauopathies characterized by Tau hyperphosphorylation, such as argyrophilic grain dementia, Pick's disease, corticobasal degeneration, progressive supranuclear palsy, and hereditary frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17) [43]. Furthermore, LRRK2 inhibitors may have utility in treating other diseases characterized by decreased dopamine levels, such as withdrawal symptoms/relapse associated with drug addiction [44].

Summary of the Invention

In one aspect, provided herein are compounds of Formula I, or stereoisomers, tautomers, N-oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts, esters, or prodrugs thereof:

in:

V is CH or N;

W is N or O;

R ¹ is absent, H, C _1-10 alkyl, C _3-10 cycloalkyl, C _2-10 heterocycloalkyl, C _6-14 aryl, C _1-10 heteroaryl, C _1-5 alkyl-C _1-10 heteroaryl, or C _1-5 alkyl-C _6-14 aryl, wherein said C _1-10 alkyl, C _3-10 cycloalkyl, C _2-10 heterocycloalkyl, C _6-14 aryl, C _1-10 heteroaryl, C _1-5 alkyl-C _1-10 heteroaryl, and C _1-5 alkyl-C _6-14 aryl are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, C _3-7 cycloalkyl, and C _2-7 heterocycloalkyl;

X ¹ is a bond, CO or -(CH ₂ ) _n -;

Y is -(CH ₂ ) _n -, -(CR ² R ³ )-, C _6-14 aryl, or C _1-10 heteroaryl, optionally R ² and R ^3, together with the carbon atom to which they are attached, form a C ₃ -C ₁₀ carbocycle or a 3- to 10-membered heterocycle, wherein said -(CH ₂ ) _n -, -(CR ² R ³ )-, C _6-14 aryl, C _1-10 heteroaryl, C ₃ -C ₁₀ carbocycle, and 3- to 10-membered heterocycle are each independently and optionally substituted with one or more substituents selected from the group consisting of C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, and C _1-6 haloalkyl;

Z is a bond, NR ² , -(CH ₂ ) _n -, or -(CR ² R ³ )-, wherein said NR ² , -(CH ₂ ) _n -, and -(CR ² R ³ )- are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl;

n is 0, 1, 2, 3, 4, or 5;

R ² and R ³ are independently selected from -H, C _1-6 alkyl, C _3-7 cycloalkyl, C _2-7 heterocycloalkyl, C _6-14 aryl or C _1-10 heteroaryl, wherein the C _1-6 alkyl, C _3-7 cycloalkyl, C _2-7 heterocycloalkyl, C _6-14 aryl and C _1-10 heteroaryl are each optionally and independently substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, and -CO ₂ H.

In some embodiments, R ¹ is H, C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _6-10 aryl, C _3-8 heteroaryl, C _1-3 alkyl-C _1-7 heteroaryl, or C _1-3 alkyl-C _6-10 aryl, wherein the C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _6-10 aryl, C _3-8 heteroaryl, C _1-3 alkyl-C _1-7 heteroaryl, and C _1-3 alkyl-C _6-10 aryl are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, C _3-7 cycloalkyl, and C _3-7 heterocycloalkyl.

In some embodiments, X ¹ is CO, -CH ₂ -, -(CH ₂ ) ₂ -, or -(CH ₂ ) ₃ -.

In some embodiments, Y is -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CR ² R ³ )-, C _6-10 aryl, or C _3-8 heteroaryl, optionally R ² and R ³ , together with the carbon atom to which they are attached, form a C ₃ -C ₈ carbocycle or a 3- to 8-membered heterocycle, wherein said -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CR ² R ³ )-, C _6-10 aryl, C _3-8 heteroaryl, C ₃ -C ₈ carbocycle, and 3- to 8-membered heterocycle are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl.

In some embodiments, Y is -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CR ² R ³ )-,

wherein Q ₁ , Q ₂ , Q ₃ and Q ₄ are each independently C or N, and Q ₁ , Q ₂ , Q ₃ and Q ₄ are each optionally substituted by one or more substituents selected from C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl and C _1-6 haloalkyl,

wherein Q ₅ , Q ₆ and Q ₇ are each independently C, N, O or S, and Q ₅ , Q ₆ and Q ₇ are each optionally substituted by one or more substituents selected from C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl or C _1-6 haloalkyl.

In some embodiments, Z is a bond, NR ² , -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, or -(CR ² R ³ )-, wherein said NR ² , -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, and -(CR ² R ³ )- are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl.

In some embodiments, R ² and R ³ are independently selected from -H, C _1-3 -alkyl, C _3-5 cycloalkyl, C _3-5 heterocycloalkyl, C _6-8 aryl, or C _3-8 heteroaryl, wherein the C _1-3 alkyl, C _{3-5 cycloalkyl, C 3-5 heterocycloalkyl} , C _6-8 aryl, and C _3-8 heteroaryl are each optionally and independently substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, and -CO ₂ H.

In some embodiments, R ¹ is selected from the following groups:

In some embodiments, R ¹ is preferably selected from the following groups:

In some embodiments, Y is -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CR ² R ³ )-, a benzene ring, a 5- to 6-membered heteroaryl ring, a C _6-10 aryl group, or a C _3-8 heteroaryl group, optionally R ² and R ^3, together with the carbon atom to which they are attached, form a C ₃ -C ₆ carbocycle or a 3- to 6-membered heterocycle, wherein the benzene ring, the 5- to 6-membered heteroaryl ring, the C _6-10 aryl group, the C _3-8 heteroaryl group, the C ₃ -C ₆ carbocycle, and the 3- to 6-membered heterocycle are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, methyl, ethyl, n-propyl, isopropyl, -CF ₃ , and C _1-3 haloalkyl.

In some embodiments, Y is selected from the following groups:

In some embodiments, Y is preferably selected from the following groups:

In some embodiments, provided herein are compounds having one of the following structures, or a stereoisomer, tautomer, N-oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt, ester, or prodrug thereof:

In some embodiments, provided herein are compounds preferably having one of the following structures, or a stereoisomer, tautomer, N-oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt, ester, or prodrug thereof:

In another aspect, provided herein are pharmaceutical compositions comprising a compound disclosed herein.

In some embodiments, the pharmaceutical composition disclosed herein further comprises a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof.

In some embodiments, the pharmaceutical compositions disclosed herein further comprise a second therapeutic agent.

In another aspect, provided herein is a use of a compound or pharmaceutical composition disclosed herein in the preparation of a medicament for treating a condition caused by at least one of cancer and a neurodegenerative disease.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein are methods of treating a condition caused by at least one of cancer and a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of a compound or pharmaceutical composition disclosed herein.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein are compounds or pharmaceutical compositions disclosed herein for use in treating a condition caused by at least one of cancer and a neurodegenerative disease.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein is the use of a compound or pharmaceutical composition disclosed herein in the preparation of a medicament for preventing or treating a condition caused by, associated with, or associated with any aberrant kinase activity.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein are methods for preventing or treating a condition caused by, associated with, or associated with any aberrant kinase activity, comprising administering to a subject a therapeutically effective amount of a compound or pharmaceutical composition disclosed herein.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein are compounds or pharmaceutical compositions disclosed herein for use in preventing or treating a condition caused by, associated with, or associated with any aberrant kinase activity.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein is the use of a compound or pharmaceutical composition disclosed herein in an assay for identifying additional candidate compounds capable of inhibiting a kinase.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein is a compound of Formula II, or a stereoisomer, tautomer, N-oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt, ester, or prodrug thereof:

Wherein V is CH or N;

W is N or O;

R ¹ is absent, H, C _1-10 alkyl, C _3-10 cycloalkyl, C _2-10 heterocycloalkyl, C _6-14 aryl, C _1-10 heteroaryl, C _1-5 alkyl-C _1-10 -heteroaryl, or C _1-5 -alkyl-C _6-14 -aryl, wherein said C _1-10 alkyl, C _3-10 cycloalkyl, C _2-10 heterocycloalkyl, C _6-14 aryl, C _1-10 heteroaryl, C _1-5 alkyl-C _1-10 -heteroaryl and C _1-5 -alkyl-C _6-14 -aryl are each independently and optionally selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, C _3-7 cycloalkyl, C substituted with one or more substituents from _{2 to 7} heterocycloalkyl, amide, sulfonamide, and sulfone;

X ¹ is a bond, CO, or -(CH ₂ ) _n -;

Y is -(CH ₂ ) _n -;

Z is a bond, N, or -(CH ₂ ) _n -;

R ⁴ is each independently absent, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, or C _1-6 haloalkyl, optionally two R ⁴ together with the Y to which they are attached form a C ₃ -C ₁₀ carbocycle or a 3- to 10-membered heterocycle, wherein the C ₃ -C ₁₀ carbocycle and the 3- to 10-membered heterocycle are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl;

R ⁵ is absent, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, C _1-6 haloalkyl, optionally R ⁴ and R ⁵ together with YZ to which they are attached form a phenyl ring, a C ₃ -C ₁₀ carbocycle, a 3- to 10-membered heterocycle, or a 5- to 10-membered heteroaryl ring, wherein the phenyl ring, C ₃ -C ₁₀ carbocycle, 3- to 10-membered heterocycle, and 5- to 10-membered heteroaryl ring are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl;

k is 0, 1, 2, 3, or 4;

n is 0, 1, 2, 3, 4, or 5;

In some embodiments, R ¹ is H, C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _6-10 aryl, C _1-8 heteroaryl, C _1-3 alkyl-C _1-8 -heteroaryl, or C _1-3 -alkyl-C _6-10 -aryl, wherein the C _1-6 alkyl, C _3-7 cycloalkyl, C _3-7 heterocycloalkyl, C _6-10 aryl, C _1-8 heteroaryl, C _1-3 alkyl-C _1-8 -heteroaryl, and C _1-3 -alkyl-C _6-10 -aryl are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, C _3-7 cycloalkyl, and C _3-7 heterocycloalkyl.

In some embodiments, X ¹ is CO, -CH ₂ -, -(CH ₂ ) ₂ -, or -(CH ₂ ) ₃ -.

In some embodiments, Y is _-CH2- , -( _CH2 ) _2- , or -( _CH2 ) _3- .

In some embodiments, Z is a bond, N, _-CH2- , -( _CH2 ) _2- , or -( _CH2 ) _3- .

In some embodiments, each R ⁴ is independently absent, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-3 alkyl, or C _1-3 haloalkyl, and optionally two R ⁴ connected to Y are taken together with Y to form a C ₃ -C ₇ carbocycle or a 3 to 7-membered heterocycle, wherein the C ₃ -C ₇ carbocycle and the 3 to 7-membered heterocycle are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, and C _1-6 haloalkyl.

In some embodiments, R ⁵ is absent, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-3 alkyl, or C _1-6 haloalkyl, optionally R ⁴ and R ⁵ together with YZ to which they are attached form a phenyl ring, a C ₃ -C ₇ carbocycle, a 3 to 7 membered heterocycle, or a 5 to 7 membered heteroaryl ring, wherein the phenyl ring, C ₃ -C ₇ carbocycle, 3 to 7 membered heterocycle, and 5 to 7 membered heteroaryl ring are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-6 alkyl, or C _1-6 haloalkyl.

In some embodiments, R ¹ is selected from the following groups:

In some embodiments, R ⁵ is absent, F, Cl, Br, I, -NO ₂ , -CN, -N ₃ , -NH ₂ , -OH, C _1-3 alkyl, or C _1-3 haloalkyl, optionally R ⁴ and R ⁵ together with YZ to which they are attached form a phenyl ring or a pyrazole ring, wherein the phenyl ring and the pyrazole ring are each independently and optionally substituted with one or more substituents selected from F, Cl, Br, -CN, -OH, -CO ₂ H, and -CF ₃ .

In another aspect, provided herein are pharmaceutical compositions comprising a compound of Formula II disclosed herein.

In some embodiments, the pharmaceutical composition disclosed herein further comprises a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof.

In some embodiments, the pharmaceutical compositions disclosed herein further comprise a second therapeutic agent.

In another aspect, provided herein is a use of a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein in the preparation of a medicament for treating a condition caused by at least one of cancer and a neurodegenerative disease.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein is a method for treating a disease caused by at least one of cancer and a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein are compounds of Formula II or pharmaceutical compositions comprising compounds of Formula II disclosed herein for use in treating a condition caused by at least one of cancer and a neurodegenerative disease.

In some embodiments, the neurodegenerative disease is Parkinson's disease.

In another aspect, provided herein is the use of a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein in the preparation of a medicament for preventing or treating a condition caused by, associated with, or associated with any abnormal kinase activity.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein is a method for preventing or treating a condition caused by, associated with, or associated with any aberrant kinase activity, comprising administering to a subject a therapeutically effective amount of a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein are compounds of Formula II or pharmaceutical compositions comprising compounds of Formula II disclosed herein for use in preventing or treating a condition caused by, associated with, or associated with any aberrant kinase activity.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

In another aspect, provided herein is the use of a compound of Formula II or a pharmaceutical composition comprising a compound of Formula II disclosed herein in an assay for identifying additional candidate compounds capable of inhibiting kinases.

In some embodiments, the kinase is LRRK.

In some embodiments, the kinase is LRRK2.

DETAILED DESCRIPTION

Definitions and General Terms

All references cited in the present invention are incorporated herein by reference in their entirety, and in the event of inconsistencies between the references introduced and the present invention, the present disclosure shall prevail. In addition, all terms and phrases used herein have the ordinary meanings known to those skilled in the art. Even so, it is still necessary to describe the terms and phrases of the present invention in more detail. In the event of inconsistencies between the terms and phrases mentioned and the meanings known, the present disclosure shall prevail. Regardless of whether the terms in question appear individually or in combination, the following definitions of the conventional terms used in this specification sheet apply.

As used herein, the grammatical articles "a," "an," and "the" are intended to include "at least one" or "more than one," unless otherwise indicated herein or clearly contradicted by context. Thus, the articles used herein refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "component" refers to more than one component, and thus, more than one component is contemplated and may be employed or used in the implementation of the described embodiments.

As described herein, the compounds disclosed herein may be optionally substituted with one or more substituents, as exemplified generally below, or as exemplified by particular classes, subclasses, and species of the invention.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

The term "alkyl" refers to a saturated, linear or branched, monovalent hydrocarbon radical of 1 to 10 carbon atoms. Unless otherwise specified, an alkyl group contains 1-10 carbon atoms. In some embodiments, the alkyl group contains 1-8 carbon atoms; in other embodiments, the alkyl group contains 1-6 carbon atoms; in yet other embodiments, the alkyl group contains 1-4 carbon atoms; and in yet other embodiments, the alkyl group contains 1-3 carbon atoms. Alkyl groups are optionally substituted with one or more substituents described herein.

Some non-limiting examples _of alkyl groups include methyl (Me, _-CH3 ), ethyl (Et, _-CH2CH3 ), n _- propyl (n-Pr _{, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2 ) , n -} butyl (n-Bu _{, -CH2CH2CH2CH3} ), isobutyl (i-Bu, _{-CH2CH (} CH3) ₂ ) _, sec-butyl (s-Bu, -CH( _CH3 ) _CH2CH3 ), tert-butyl (t-Bu, -C( _{CH3 ) 3} ) _, n-pentyl ( _{-CH2CH2CH2CH2CH3 ) , 2} -pentyl ( _-CH ( _CH3 ) _CH2CH2CH3 ), 3-pentyl (-CH( _{CH2CH3 ) 2} ) _{, n} -hexyl ( _{-CH2CH2CH2CH3} ) _{, 2} CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), etc.

The term "cycloalkyl" refers to a monovalent or multivalent saturated ring having 3 to 10 carbon atoms, such as a monocyclic, bicyclic or tricyclic ring system. The bicyclic or tricyclic ring system may include fused rings, bridged rings, and spirocycles. In some embodiments, the cycloalkyl group contains 3 to 8 carbon atoms. In other embodiments, the cycloalkyl group contains 3 to 6 carbon atoms. A cycloalkyl radical is optionally substituted with one or more substituents described herein.

The term "aryl" refers to a monovalent or polyvalent monocyclic, bicyclic or tricyclic carbocyclic ring system having a total of 6 to 12 ring members, preferably 6 to 10 ring members, and more preferably 6 ring members, and wherein at least one ring in the system is aromatic. The aryl group is usually, but not necessarily, bound to the parent molecule through the aromatic ring of the aryl group. The terms "aryl" and "aromatic ring" can be used interchangeably herein. Examples of aryl groups can include phenyl, naphthyl, anthracene, etc. Aryl radicals are optionally substituted with one or more substituents described herein.

The term "heteroaryl" refers to a monovalent or multivalent monocyclic, bicyclic or tricyclic ring system having a total of 5 to 10 ring members, and wherein at least one ring in the system is aromatic and at least one ring contains one or more heteroatoms. The heteroaryl group is typically, but not necessarily, bound to the parent molecule through the aromatic ring of the heteroaryl group. The terms "heteroaryl" and "heteroaromatic ring" or "heteroaromatic compound" are used interchangeably herein. The heteroaryl group is optionally substituted with one or more substituents disclosed herein. In some embodiments, the 5- to 10-membered heteroaryl group contains 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N; in some other embodiments, the 5- to 6-membered heteroaryl group is a monocyclic ring system and contains 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N.

Some non-limiting examples of heteroaryl rings include thienyl, furanyl, pyrrolyl, pyridinyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, tetrazolyl and the like and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzimidazolyl, indolyl, isoindolyl, indazolyl and the like; or pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like and benzo derivatives thereof, such as quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl and the like.

"Heterocycloalkyl" refers to a cyclic aliphatic group containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur, which is optionally interrupted by one or more -(CO)- groups in the ring and/or optionally contains one or more double bonds in the ring. Alternatively, the heterocycloalkyl group is _C4-7 -heterocycloalkyl, more preferably _C4-6 -heterocycloalkyl. Preferred heterocycloalkyl groups include, but are not limited to, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl, tetrahydrofuranyl, and tetrahydropyranyl.

Description of the compounds of the present invention

Therapeutic applications

Another aspect of the present invention relates to the above-mentioned compounds for use in medicine.

Another aspect of the present invention relates to the above compounds for use in the treatment of cancer or neurodegenerative diseases.

Another aspect relates to the use of the above compound in the preparation of a medicament for treating or preventing a neurodegenerative disease. Preferably, the neurodegenerative disease is Parkinson's disease.

Another aspect relates to the use of the above compounds in the preparation of a medicament for treating or preventing a proliferative disease, such as cancer.

Preferably, the compound is administered in an amount sufficient to inhibit more than one kinase, preferably LRRK, even more preferably LRRK2.

Yet another aspect relates to the use of a compound of the present invention in the preparation of a medicament for preventing or treating a condition caused by, associated with or associated with any abnormal activity against a biological target, wherein the target is a kinase, more preferably LRRK, even more preferably LRRK2.

Preferably, the disorder is Parkinson's disease.

Another aspect of the present invention relates to a method for treating a protein kinase-related disease or condition. The method according to this aspect of the invention is achieved by administering to a subject in need thereof a therapeutically effective amount of a compound of the invention as described above, either per se, or more preferably as part of a pharmaceutical composition mixed with a pharmaceutically acceptable carrier, such as described in detail below.

Yet another aspect of the present invention relates to a method of treating a mammal suffering from a disease state alleviated by inhibition of a protein kinase, wherein the method comprises administering to the mammal a therapeutically effective amount of a compound of the present invention.

Preferably, the disease state is alleviated by inhibiting the protein kinase LRRK, more preferably LRRK2.

Preferably, the mammal is a human.

The term "method" refers to ways, means, techniques and procedures for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to or readily developable by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine from known ways, means, techniques and procedures.

As used herein, the term "administering" refers to a method of combining a compound of the present invention with a protein kinase in such a way that the compound can affect the enzymatic activity of the protein kinase, either directly, i.e., by interacting with the protein kinase itself, or indirectly, i.e., by interacting with other molecules on which the catalytic activity of the protein kinase is dependent. As used herein, administration can be accomplished in vitro (i.e., in a test tube) or in vivo (i.e., in cells or tissues of a living organism).

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease or condition, substantially ameliorating the clinical symptoms of a disease or condition or substantially preventing the appearance of clinical symptoms of a disease or condition.

As used herein, the term "prevent" or "preventing" refers to a method of preventing an organism from acquiring a condition or disease in the first place.

The term "therapeutically effective amount" means that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated.

For any compound used in the present invention, a therapeutically effective amount (also referred to herein as a therapeutically effective dose) can be initially estimated from cell culture assays. For example, a dose can be formulated to achieve a circulating concentration range in an animal model that includes the _IC50 or _IC100 determined in cell culture. This information can be used to more accurately determine a useful dose in humans. An initial dose can also be estimated from in vivo data. Using these preliminary guidelines, one of ordinary skill in the art can determine an effective dose in humans.

In addition, the toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD ₅₀ and ED _50. The dose ratio between toxicity and therapeutic effect is a therapeutic index and can be expressed as the ratio between LD ₅₀ and ED _50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range that is nontoxic for use in humans. The dosage of these compounds preferably lies within a range that includes circulating concentrations with little or no toxicity at ED _50. The dosage can vary within this range depending on the dosage form used and the route of administration adopted. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

Dosage and interval can be adjusted separately to provide the plasma level of the active compound that is enough to maintain therapeutic effect.The scope of conventional patient dosage for oral administration is about 1-2000mg/kg/day, generally about 2-1000mg/kg/day, preferably about 5-700mg/kg/day and most preferably about 10-500mg/kg/day.Preferably, by giving multiple dosages every day, realize the effective serum level of treatment.In the case of topical administration or selective uptake, the effective local concentration of medicine may be unrelated to plasma concentration.Those skilled in the art will be able to optimize the effective local dose for treatment without excessive experiment.

As used herein, a "kinase-associated disease or condition" refers to a disease or condition characterized by inappropriate kinase activity or excessive activity of a kinase as defined herein. Inappropriate activity refers to (i) expression of a kinase in cells that do not normally express the kinase; (ii) increased expression of a kinase, resulting in unwanted cell proliferation, differentiation and/or growth; or (iii) decreased expression of a kinase, resulting in an undesirable reduction in cell proliferation, differentiation and/or growth. Excessive activity of a kinase refers to the amplification of a gene encoding a particular kinase or the generation of a certain level of kinase activity, which can be associated with a cell proliferation, differentiation and/or growth disorder (i.e., as the level of the kinase increases, the severity of the symptoms of one or more cell disorders increases). Excessive activity can also be the result of ligand-independent or constitutive activation due to a mutation, such as a deletion of a fragment of the kinase responsible for ligand binding.

Preferred diseases or conditions that the compounds described herein can be used to prevent include cancer and neurodegenerative diseases, such as Parkinson's disease.

Therefore, the present invention also provides the use of a compound as defined herein for the preparation of a medicament for the treatment of a disease for which inhibition of LRRK2 is desired, including Parkinson's disease.

Pharmaceutical composition

For use according to the present invention, the compounds described herein, or their physiologically acceptable salts, esters, or other physiologically functional derivatives, may be presented as pharmaceutical preparations comprising the compounds, or their physiologically acceptable salts, esters, or other physiologically functional derivatives, and one or more pharmaceutically acceptable carriers, and optionally other therapeutic and/or prophylactic ingredients. The carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. Pharmaceutical compositions can be used for human or animal use in both human and veterinary medicine.

Examples of suitable excipients for use in the various forms of pharmaceutical compositions described herein may be found in "Handbook of Pharmaceutical Excipients, 2nd edition, (1994), edited by A Wade and PJ Weller".

Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, adjuvant or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain or additionally contain any suitable adhesive, lubricant, suspending agent, coating agent, solubilizer, buffer, flavoring, surfactant, thickener, preservative (including antioxidant) etc. as a carrier, adjuvant or diluent, as well as the substance contained in order to make the preparation isotonic with the blood of the intended recipient.

Examples of suitable binders include starch, gelatin, natural sugars (such as glucose, anhydrous lactose, free lactose, beta-lactose), corn sweeteners, natural and synthetic gums (such as acacia, tragacanth) or sodium alginate, carboxymethylcellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.

Preservatives, stabilizers, dyes, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), intranasal and pulmonary administration (e.g., by inhalation). The formulations may be conveniently presented in discrete dosage units where appropriate and may be prepared by any method well known in the pharmaceutical art. All methods include the step of bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration in which the carrier is a solid are most preferably in the form of unit dose formulations, such as pills, capsules, or tablets, each containing a predetermined amount of the active compound. Tablets can be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing the active compound in a free-flowing form (such as a powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, lubricating substance, surfactant, or dispersant. Molded tablets can be prepared by molding the active compound and an inert liquid diluent. The tablets can optionally be coated, or, if not coated, can optionally be printed with a symbol. Capsules can be prepared by filling the active compound, alone or in admixture with one or more auxiliary ingredients, into a capsule shell and then sealing it in a conventional manner. Cachets are similar to capsules, in which the active compound, along with any auxiliary ingredients, is sealed in rice paper film. The active compound can also be formulated as dispersible granules, which can, for example, be suspended in water or sprinkled on food before administration. The granules can be packaged, for example, in sachets. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosage forms, such as tablets, in which the active compound is formulated in a suitable controlled release matrix or coated with a suitable controlled release film. Such formulations are particularly convenient for prophylactic use.

Pharmaceutical preparations suitable for rectal administration (wherein the carrier is a solid) are most preferably presented in the form of unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be easily formed by mixing the active compound with a softened or melted carrier, then cooling and molding in a mold. Pharmaceutical preparations suitable for parenteral administration include sterile solutions or suspensions of the active compound in aqueous or oily solvents.

Injectable formulations can be suitable for bolus injection or continuous injection. Such formulations are conveniently presented in unit dose or multidose containers, which are sealed after the formulation is introduced until needed for use. Alternatively, the active compound can be in powder form, which is formed with a suitable solvent, such as sterile pyrogen-free water, before use.

The active compound can also be formulated into a long-acting, lasting formulation that can be administered intramuscularly or by implantation, for example, subcutaneously or intramuscularly. Long-lasting formulations can include, for example, suitable polymers or hydrophobic materials or ion exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the oral cavity are presented such that particles containing the active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered to the recipient's bronchial tree.

As one possibility, such a formulation is in the form of a finely divided powder which can conveniently be presented in a pierceable capsule suitable for example for gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising the active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations in which the active compound is dispensed in the form of droplets of a solution or suspension may also be employed.

Such self-propelling formulations are similar to those known in the art and can be prepared by established methods. Suitably, they are presented in a container provided with a manually operated or automatically functioning valve having the desired spray characteristics; advantageously, they are of a metered type that delivers a fixed volume, for example 25 to 100 microliters, each time the valve is operated.

As a further possibility, the active compound may be in the form of a solution or suspension for use in a nebulizer or sprayer, whereby accelerating air flow or ultrasonic agitation is employed to produce a mist of fine droplets for inhalation.

Formulations suitable for intranasal administration include those generally similar to those described above for pulmonary administration. Such formulations should desirably have a particle size in the range of 10 to 200 microns to enable retention in the nasal cavity when the formulation is dispensed; this can be achieved by appropriately using a powder of the appropriate particle size or selecting an appropriate valve. Other suitable formulations include coarse powders with a particle size in the range of 20-500 microns for administration by rapid inhalation through the nasal passages from a container held close to the nose, and nasal drops containing 0.2 to 5% w/v of the active compound in an aqueous or oily solution or suspension.

Pharmaceutically acceptable carriers are well known to those skilled in the art, and include but are not limited to 0.1M and preferably 0.05M phosphate buffer or 0.8% saturated common salt water. In addition, these pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saturated common salt water and buffered media. Parenteral solvents include sodium chloride solution, Ringer's dextrose (Ringer's dextrose), glucose and sodium chloride, lactated Ringer's solution (lactated Ringer's) or fixed oils. Preservatives and other additives may also be present, for example, such as antimicrobials, antioxidants, chelating agents, inert gases, etc.

Formulations suitable for topical preparations may be provided, such as gels, creams or ointments. Such formulations may be applied, for example, to a wound or ulcer, either directly on the surface of the wound or ulcer, or carried on a suitable support, such as a bandage, gauze, mesh, etc., which may be applied over the area to be treated.

Liquid or powder formulations can also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g., a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh, etc. can be sprayed or sprinkled with the formulation and then applied to the site to be treated.

According to another aspect of the present invention there is provided a process for preparing a pharmaceutical or veterinary composition as described above, which process comprises bringing into association the active compound with the carrier, for example by mixing.

In general, the formulations are prepared by uniformly and intimately combining the active agent with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product. The present invention relates to a process for preparing a pharmaceutical composition comprising combining or associating a compound of formula (I) with a pharmaceutically or veterinarily acceptable carrier or solvent.

Salt/Ester

The compounds of the present invention may be present as salts or esters, in particular pharmaceutically and veterinarily acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the present invention include suitable acid addition salts or base salts thereof. A review of suitable pharmaceutical salts can be found in Berge et al., J Pharm Sci, 66, 199 (1977). Salts are formed, for example, with strong inorganic acids, such as mineral acids, for example hydrohalic acids (e.g., hydrochloric acid, hydrobromic acid and hydroiodic acid), sulfuric acid, phosphosulfates, hydrogensulfates, hemisulfates, thiocyanates, persulfates and sulfonic acids; with strong organic carboxylic acids, for example unsubstituted or substituted (e.g., by halogen) alkanecarboxylic acids having 1 to 4 carbon atoms, such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; with hydroxycarboxylic acids, for example, ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; with amino acids, for example, aspartic acid or glutamic acid; with benzoic acid; or with organic sulfonic acids, for example unsubstituted or substituted (e.g., by halogen) (C ₁ -C ₄ )-alkyl- or aryl-sulfonic acids, such as methane- or p-toluenesulfonic acid. Salts that are not pharmaceutically or veterinarily acceptable may still be valuable intermediates.

Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanoate, glucoheptonate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmitate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulfonic acids such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, camphorsulfonate, 2-naphthalenesulfonate, benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; and inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, hydrogensulfate, hemisulfuric acid, thiocyanic acid, persulfuric acid, phosphoric acid and sulfonic acid.

Depending on the functional group being esterified, esters are formed using organic acids or alcohols/hydroxides. Organic acids include carboxylic acids such as unsubstituted or substituted (e.g., by halogen) alkanecarboxylic acids having 1 to 12 carbon atoms, such as acetic acid; saturated or unsaturated dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or tetraphthalic acid; hydroxycarboxylic acids such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid; amino acids such as aspartic acid or glutamic acid; benzoic acid; or organic sulfonic acids such as unsubstituted or substituted (e.g., by halogen) (C ₁ -C ₄ )-alkyl- or aryl-sulfonic acids such as methane- or p-toluenesulfonic acid. Suitable hydroxides include inorganic hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide. Alcohols include alkanols which may be unsubstituted or substituted (e.g., by halogen) 1 to 12 carbon atoms.

Enantiomers/tautomers

In all aspects of the invention previously discussed, where appropriate, the invention includes all enantiomers, diastereomers, and tautomers of the compounds of the invention. One skilled in the art will recognize compounds having optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers can be separated/prepared by methods known in the art.

Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold, and Prelog. These conventions are well known in the art (e.g., see "Advanced Organic Chemistry," 3rd edition, March, J., John Wiley and Sons, New York, 1985).

Compounds of the present invention containing a chiral center can be used as racemic mixtures, enantiomerically enriched mixtures, or the racemic mixtures can be separated using well-known techniques and the individual enantiomers used individually.

Stereoisomers and geometric isomers

Some of the compounds of the present invention may exist as stereoisomers and/or geometric isomers - for example, they may have more than one asymmetric and/or geometric center and, therefore, may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all of these inhibitors as individual stereoisomers and geometric isomers, as well as mixtures thereof. The terms used in the claims encompass these forms, so long as the form retains the appropriate functional activity (although not necessarily to the same extent).

The present invention also includes all suitable isotopic variants of medicament or its pharmaceutically acceptable salt.An isotopic variant of the medicament of the present invention or its pharmaceutically acceptable salt is defined as a kind of atom wherein at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass of the atom usually found in nature. The isotopic example that can be incorporated into this medicament and its pharmaceutically acceptable salt includes the isotope of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine, such as respectively ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. Certain isotopic variants of medicament and pharmaceutically acceptable salt, for example, wherein incorporate as ³ H or ¹⁴ those isotopic variants of the radioisotope of C, can be used for drug and/or substrate tissue distribution studies. Tritiated, i.e. ³ H, and carbon-14, i.e. ¹⁴ C isotopes, are particularly preferred due to their ease of preparation and detectability. In addition, substitution with isotopes such as deuterium (i.e. ² H) may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in certain cases. For example, the present invention includes compounds of formula (I) in which any hydrogen atom is replaced by a deuterium atom. Isotopic variants of the medicament of the present invention and pharmaceutically acceptable salts thereof of the present invention can generally be prepared by conventional methods using appropriate isotopic variants of suitable reagents.

Prodrug

The present invention also includes the compounds of this invention in the form of prodrugs, i.e., compounds that release the covalent bond of the active parent drug according to general formula (I) in vivo. Such prodrugs are typically compounds of this invention in which one or more appropriate groups have been modified so that the modification may be reversed after being administered to a human or mammalian subject. This reversal is usually carried out by a naturally occurring enzyme in this type of subject, although a second agent may be administered together with this prodrug to reverse in vivo. Examples of such modifications include esters (e.g., any of the above), which can be reversed by esterases etc. Other such systems are well known to those skilled in the art.

Solvates

The present invention also includes solvated forms of the compounds of the present invention. The terms used in the claims include these forms.

Polymorph

The present invention also relates to the compounds of the present invention in various crystalline forms, polymorphic forms and hydrated forms of the compounds of the present invention. It has been determined in the pharmaceutical industry that chemical compounds can be isolated in any of these forms by slightly changing the methods of purification and/or separation of the solvents used in the synthetic preparation of such compounds.

Drug administration

The pharmaceutical composition of the present invention is applicable to rectal, intranasal, intrabronchial, local (including oral and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or intrathecal administration. Preferably, the preparation is a preparation for oral administration. The preparation can be conveniently presented in unit dosage form, i.e., in the form of discrete portions of multiple units or subunits comprising a unit dose or a unit dose. As an example, the preparation can be in the form of tablets and sustained-release capsules, and can be prepared by any method known to the pharmaceutical field.

The formulations of the present invention for oral administration can be presented as discrete units such as capsules, gels, drops, cachets, pills or tablets, each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion or suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus injection, etc. Preferably, each dose of these compositions contains 1 to 250 mg and more preferably 10-100 mg of the active ingredient.

For compositions for oral administration (e.g., tablets and capsules), the term "acceptable carrier" includes solvents such as conventional excipients, for example, binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose, and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; and lubricants such as magnesium stearate, sodium stearate, and other metal stearates, glyceryl stearate, stearic acid, silicone fluid, talc, oil, and colloidal silicon dioxide. Flavoring agents such as peppermint, wintergreen oil, cherry flavor, etc. may also be used. It may be necessary to add a colorant to make the dosage form easily recognizable. Tablets may also be coated by methods well known in the art.

Tablets can be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing an activating agent in a free-flowing form (such as powder or granule) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, lubricating substance, preservative, surfactant or dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide a slow or controlled release of an activating agent.

Other formulations suitable for oral administration include lozenges containing the active agent in a flavored basis, usually sucrose and acacia or tragacanth; pastilles containing the active agent in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

Other administration forms include solutions or emulsions that can be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly and prepared from sterile or sterilizable solutions. Injectable forms usually contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.

The pharmaceutical composition of the present invention may also be in the form of anal suppositories, vaginal suppositories, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dustings.

Another method of transdermal administration is the use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active ingredient can also be incorporated into an ointment consisting of white wax or white soft paraffin, and optionally stabilizers and preservatives, at a concentration of 1 to 10% by weight.

dose

Those of ordinary skill in the art can easily determine the appropriate dosage of one of the compositions of the present invention to be administered to a subject without excessive experimentation. Typically, a physician will determine the actual dosage that is most suitable for an individual patient, and the dosage will depend on a variety of factors, including the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health status, sex, diet, mode of administration and time, excretion rate, drug combination, the severity of a specific condition, and the individual being treated. The dosages disclosed herein are examples of average conditions. Of course, there may be individual cases in which the higher or lower dosage range is valuable, and this dosage range is within the scope of the present invention.

According to the present invention, an effective amount of a compound of formula (I) can be administered to inhibit a kinase associated with a particular condition or disease. Of course, the dosage will be further modified depending on the type of administration of the compound. For example, it is preferred that a compound of formula (I) be administered parenterally to achieve an "effective amount" for acute treatment. Intravenous infusion of the compound in a 5% aqueous dextrose solution or saline, or a similar formulation with suitable excipients, is most effective, although intramuscular bolus injection is also useful. Typically, the parenteral dose is from about 0.01 to about 100 mg/kg; preferably, between 0.1 and 20 mg/kg, in such a way as to maintain plasma drug concentrations at levels effective for kinase inhibition. The compound can be administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 400 mg/kg/day. One of ordinary skill in the art can readily determine the precise amount of a therapeutically effective compound of the invention, as well as the optimal route of administration of the compound, by comparing blood levels of the agent to the concentration required for therapeutic efficacy.

The compounds of the present invention may also be administered orally to a patient in a manner that provides a drug concentration sufficient to treat one or more of the therapeutic indications disclosed herein. Typically, a pharmaceutical composition containing the compound is administered orally in a manner consistent with the patient's condition at a dose of between about 0.1 and about 50 mg/kg. Preferably, the oral dose is between about 0.5 and about 20 mg/kg.

When the compounds of the invention are administered according to the present invention, no unacceptable toxicological effects are expected.Compounds of the invention that may have good bioavailability may be tested in one of a variety of bioassays to determine the concentration of compound required to have a given pharmacological effect.

combination

In a particularly preferred embodiment, one or more compounds of the present invention are administered in combination with one or more other active agents (e.g., existing drugs available on the market). In this case, the compounds of the present invention can be administered continuously, simultaneously or sequentially with one or more other active agents.

In general, drugs are more effective when used in combination. In particular, combination therapy is desirable to avoid overlapping of major toxicities, mechanisms of action, and resistance mechanisms. In addition, it is also desirable to administer most drugs with the minimum time interval between the maximum tolerated dose and this dose. The major advantage of combining chemotherapeutic drugs is that they can promote additional or possible synergistic effects through biochemical interactions and may also reduce the occurrence of drug resistance.

Beneficial combinations can be proposed by studying the inhibitory activity of a test compound with an agent known or suspected to be valuable in treating a particular condition. This method can also be used to determine the order in which such agents should be administered, i.e., before, simultaneously with, or after the delivery of the compound. This mode of administration can be a feature of all active agents identified herein.

test

Another aspect of the invention relates to the use of a compound as described above in an assay for identifying further candidate compounds capable of inhibiting more than one kinase, more preferably LRRK, even more preferably LRRK2.

Preferably, the assay is a competitive binding assay.

More preferably, the competitive binding assay comprises contacting a compound of the invention with a kinase, preferably LRRK, more preferably LRRK2, and a candidate compound, and detecting any changes in the interaction between the compound according to the invention and the kinase.

Preferably, candidate compounds are generated by conventional SAR modification of compounds of the invention.

As used herein, the term "conventional SAR modification" refers to standard methods known in the art for altering a given compound by chemical derivatization.

Therefore, in one aspect, the identified compound can be used as a model (e.g., template) for developing other compounds. The compound used in this test can be free in solution, fixed on a solid support, carried on a cell surface, or located within a cell. The activity elimination or formation of a binding complex between the compound and the agent to be tested can be measured.

The assays of the invention may be screening, whereby a large number of agents are tested. In one aspect, the assays of the invention are high throughput screening.

The present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding to a compound specifically compete with a test compound for binding to the compound.

Another technique for screening is provided for high throughput screening (HTS) of agents having suitable binding affinity for a substance and is based on the method described in detail in WO 84/03564.

It is expected that the assays of the present invention will be suitable for both small-scale and large-scale screening of test compounds as well as for quantitative assays.

Preferably, a competitive binding assay comprises contacting a compound of the invention with a kinase in the presence of a known substrate for the kinase and detecting any change in the interaction between the kinase and the known substrate.

Another aspect of the present invention provides a method for detecting binding of a ligand to a kinase, the method comprising the following steps:

(i) contacting a ligand with the kinase in the presence of a known substrate of the kinase;

(ii) detecting any change in the interaction between the kinase and the known substrate;

And wherein the ligand is a compound of the present invention.

One aspect of the present invention relates to a method comprising the steps of:

(a) performing the above-mentioned determination method;

(b) identifying one or more ligands capable of binding to the ligand binding domain; and

(c) preparing a certain amount of the one or more ligands.

Another aspect of the present invention provides a method comprising the steps of:

(a) performing the above-mentioned determination method;

(b) identifying one or more ligands capable of binding to the ligand binding domain; and

(c) preparing a pharmaceutical composition comprising said one or more ligands.

Another aspect of the present invention provides a method comprising the steps of:

(a) performing the above-mentioned determination method;

(b) identifying one or more ligands capable of binding to the ligand binding domain;

(c) modifying the one or more ligands capable of binding to the ligand binding domain;

(d) performing the above-mentioned determination method;

(e) optionally preparing a pharmaceutical composition comprising said one or more ligands.

The present invention also relates to ligands identified by the above method.

Another aspect of the present invention relates to a pharmaceutical composition comprising a ligand identified by the above method.

Another aspect of the present invention relates to the use of a ligand identified by the above method in the preparation of a pharmaceutical composition for treating more than one condition.

The above methods can be used to screen for ligands that act as inhibitors of more than one kinase.

The compounds of general formula (I) are useful both as laboratory tools and as therapeutic agents. In the laboratory, certain compounds of the invention can be used to establish what is often referred to as "target validation" to determine whether a known or newly discovered kinase is responsible for a critical or at least important biochemical function in the determination or progression of a disease state.

Conventional synthesis method

The present invention is described by the following examples. However, it should be understood that the present invention is not limited to these embodiments, and these examples are only used to suggest methods for practicing the present invention.

The following abbreviations are used throughout this specification:

AcOH acetic acid

AlCl ₃ aluminum chloride

_BH3 Borane

Bn benzyl

BuOH n-butanol

CuI Cuprous iodide

DCM dichloromethane

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DIEA,DIPEA N,N-Diisopropylethylamine

EA Ethyl acetate

EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

EtOH

EtOAc

Et ₃ N triethylamine

HATU 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HOBT Hydroxybenzotriazole

I ₂ iodine

IPA Isopropyl alcohol

KOAc potassium acetate

KOH Potassium hydroxide

K ₃ PO ₄ potassium phosphate

_LiAlH4 Lithium Aluminum Hydride

LiCl lithium chloride

LCMS high performance liquid chromatography-mass spectrometry

MeOH methanol

MeCN Acetonitrile

MeI iodomethane

MsCl methanesulfonyl chloride

Na ₂ CO ₃ sodium carbonate

_NaHCO3 sodium bicarbonate

Na ₂ S ₂ O ₃ sodium thiosulfate

NaOH sodium hydroxide

_NaBH4 sodium borohydride

(n-Bu) ₄ NI Tetrabutylammonium iodide

n-BuLi n-butyllithium

_NH3 ammonia

NH ₄ Cl ammonium chloride

NIS N-iodosuccinimide

NMR Nuclear Magnetic Resonance

prep-HPLC preparative high performance liquid chromatography

prep-TLC preparative thin layer chromatography

PMB p-methoxybenzyl

PMBCl p-Methoxybenzyl chloride

PPh ₃ triphenylphosphine

Pd(dppf)Cl ₂ 1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride

Pd(PPh ₃ ) ₄ -Tetrakis(triphenylphosphine)palladium(0)

Pd(OAc) ₂ -palladium(II) acetate

PE petroleum ether

rt room temperature

Sphos 2-Dicyclohexylphosphino-2',6'-dimethoxybiphenyl

t-BuOK Potassium tert-butoxide

t-BuONa Sodium tert-butoxide

TEA triethylamine

TLC thin layer chromatography

THF Tetrahydrofuran

TFA trifluoroacetic acid

Trt Trityl

UV

Option 1:

Step 1 Step 1 describes the conversion of formula A to formula B, wherein X is a halogen, preferably bromine or iodine, and LG is a leaving group, such as succinimide.

The reaction is carried out in a suitable solvent in the presence of a suitable halogenating agent, such as iodine or N-bromosuccinimide, and optionally in the presence of a base, such as potassium hydroxide.

Typical conditions (X=I), 1 equivalent of formula A, 2 equivalents of _I2 , 3.7 equivalents of KOH in 1,4-dioxane at 75°C for 4 hours.

Step 2

Step 2 describes the conversion of Formula B to Formula C, wherein PG is defined as a protecting group including but not limited to tert-butoxycarbonyl-; benzyloxycarbonyl-; benzyl-; 4-methoxybenzyl-; 2,4-dimethoxybenzyl- or trityl-; and LG is defined as a leaving group such as halogen or tert-butyl carbonate.

The reaction includes blocking the pyrazole NH with a protecting group. It will be appreciated by those skilled in the art that many protecting groups can be used for this purpose (see Greene, Theodora W. and Wuts, Peter G.M. Greene ' s Protective Groups in Organic Synthesis. 4th edition (2006)). It will also be appreciated by those skilled in the art that protecting groups can be introduced at N1 or N2, and that the ratio can be varied depending on the nature of the PG or the precise reaction conditions for expansion. The reaction conditions depend on the nature of the protecting group.

Typical conditions (PG = 4-methoxybenzyl): 1 equivalent of 4-methoxybenzyl chloride; 1 equivalent of formula B, 2 equivalents of potassium hydroxide in DMF were stirred overnight at room temperature.

Step 3

Step 3 describes the conversion of Formula C to Formula D, wherein X is halogen. The group ^R1 may optionally contain a functional group that can be manipulated at a later stage of the synthetic process using standard conditions known to those skilled in the art.

The reaction involves nucleophilic substitution of the chloro group in formula C with an amino group in a suitable solvent, optionally in the presence of a Bronsted acid. The reaction typically requires heating, either thermally or using microwave irradiation.

Typical conditions: 2.5 equivalents of amine, 1 equivalent of formula C in n-butanol are heated to 180°C for 5 hours in a sealed reactor.

Step 4

Step 4 involves converting Formula D to Formula E. X is a halogen, and preferably iodine. This reaction involves cross-coupling of a substituted vinyl ester with Formula D in the presence of a suitable transition metal catalyst and a suitable base (preferably triethylamine) and optionally additional additives (such as tetrabutylammonium iodide). Those skilled in the art typically refer to this type of conversion as a "Heck reaction."

Typical conditions: 1 equivalent of formula D, 10 equivalents of vinyl ester, 2 equivalents of tetrabutylammonium iodide, 0.2 equivalents of Pd(dppf) _Cl2 in DMF; DMF:water:triethylamine (6.25:1:1), heated to 70°C overnight.

Step 5

Step 5 involves converting Formula E to Formula F. This reaction involves hydrogenating the double bond to the corresponding saturated compound with a hydrogen source in a suitable solvent in the presence of a suitable transition metal catalyst. It may be necessary or desirable to add a protic acid (such as HCl or acetic acid) to promote the reaction. Those skilled in the art will appreciate that many different metal catalysts may be used for this type of reaction, and that it may be necessary or desirable to carry out these reactions under pressure.

Typical conditions: Formula E is treated with palladium on carbon under a hydrogen atmosphere.

Step 6

Step 6 involves converting Formula F to Formula G. This reaction involves hydrolyzing the ester to the corresponding carboxylic acid in a suitable solvent in the presence of a suitable base, such as sodium hydroxide.

Typical conditions: Formula F is treated with aqueous sodium hydroxide in methanol.

Step 7

Step 7 involves converting Formula G to Formula H. This reaction involves intramolecular cyclization to form a lactam under amide bond forming reaction conditions. Those skilled in the art will appreciate that many different amide bond forming reaction conditions can be used for this type of reaction.

Typical conditions: Formula G is treated with HATU in the presence of triethylamine in dichloromethane.

Step 8

Step 8 involves converting Formula H to Formula I. The reaction involves the removal of a protecting group from the pyrazole, and the precise conditions will vary depending on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis, 4th ed. (2006).

Typical conditions (PG is 4-methoxybenzyl): Compound H is treated with trifluoroacetic acid at 70°C overnight.

Step 9

Step 9 involves converting Formula H to Formula J. This reaction involves reducing the amide to the corresponding amine using a reducing agent such as borane. One skilled in the art will appreciate that many different reducing agents can be used for this type of reaction.

Step 10

Step 10 involves the conversion of Formula J to Formula K. The reaction involves the removal of a protecting group from the pyrazole, and the precise conditions will depend on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 4th ed. (2006).

Typical conditions (PG is 4-methoxybenzyl): Compound J is treated with trifluoroacetic acid at 70°C overnight.

Option 2:

Step 11

Step 11 describes the conversion of Formula D to Formula L, wherein X and PG are as defined above. This reaction involves the cross-coupling of the halide in Formula D with an aryl or heteroaryl boronic acid or ester in a suitable solvent in the presence of a transition metal catalyst. The reaction is typically carried out at elevated temperatures using thermal or microwave heating. An inorganic base (such as sodium carbonate) is typically added to the reaction mixture. This type of conversion is known to those skilled in the art as a "Suzuki coupling."

Typical conditions: 1 equivalent of formula D, 0.09 equivalent of Pd(dppf) _2Cl2 , 1.5 equivalents _of boronic acid (or boronic ester), 3.5 equivalents of 2M aqueous sodium carbonate in 1,4-dioxane at 90°C for 18 hours.

Step 12

Step 12 involves converting Formula L to Formula M. This reaction involves hydrolyzing the ester to the corresponding carboxylic acid with water in a suitable solvent in the presence of a suitable base, such as sodium hydroxide.

Typical conditions: Formula L is treated with aqueous sodium hydroxide in methanol.

Step 13

Step 13 involves converting Formula M to Formula N. This reaction involves intramolecular cyclization under amide bond forming reaction conditions to form a lactam. One skilled in the art will appreciate that many different amide bond forming reaction conditions can be used for this type of reaction.

Typical conditions: Formula M is treated with HATU in the presence of triethylamine in dichloromethane.

Step 14

Step 14 involves the conversion of Formula N to Formula O. This reaction involves the removal of a protecting group from a pyrazole, and the precise conditions will vary depending on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis, 4th Edition (2006).

Typical conditions (PG is 4-methoxybenzyl): Compound N is treated with trichloroacetic acid at 70°C overnight.

Step 15

Step 15 involves converting Formula N to Formula P. This reaction involves reducing an amide to the corresponding amine using a reducing agent such as borane. One skilled in the art will appreciate that many different reducing agents can be used for this type of reaction.

Step 16

Step 16 involves the conversion of Formula P to Formula Q. This reaction involves the removal of a protecting group from the pyrazole, and the precise conditions will depend on the nature of the protecting group (Greene, Theodora W. and Wuts, Peter G. M. Greene's Protective Groups in Organic Synthesis. 4th ed. (2006).

Typical conditions (PG is 4-methoxybenzyl): Compound P is treated with trifluoroacetic acid at 70°C overnight.

The present invention is further described by the following non-limiting examples.

Example

General procedures for synthesizing compounds

Chromatography

High pressure liquid chromatography was performed using equipment manufactured by Agela Technologies and monitored by a multi-wavelength UV detector. Typical mobile phases used for the separation process were PE/EA, DCM/MeOH, or water/MeCN. One skilled in the art will appreciate that it may be necessary or desirable to vary the conditions for each specific compound, for example by changing the solvent composition at the beginning or end, changing the solvent or buffer, changing the run time, changing the flow rate, and/or the chromatographic column.

Analytical methods

¹ H nuclear magnetic resonance (NMR) spectroscopy was performed at room temperature on a Bruker AV 400 spectrometer in the solvents described, unless otherwise stated. In all cases, the NMR data were consistent with the proposed structure. The characteristic chemical shifts (δ) are given in parts per million using conventional abbreviations for designating major peaks: for example, s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; br, broad. Mass spectra were recorded using an Agilent 1290 Infinity/6460 triple Quad LCMS. When thin layer chromatography (TLC) was used, it refers to silica gel TLC.

Preparation of compounds

Where the preparation of starting materials is not described, these are commercially available, known in the literature, or readily available to one skilled in the art using standard procedures. Where compounds are described as being prepared analogously to the preceding examples or intermediates, it will be understood by one skilled in the art that reaction times, equivalents of reagents, and temperatures may be varied for each particular reaction, and that different workup or purification techniques may be necessary or desirable.

Example 1

Step 1

Synthesis of 4-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine (Intermediate 1):

At room temperature, iodine (19 g, 76 mmol) was added to a mixture of 4-chloro-1H-pyrazolo[4,3-c]pyridine (5.8 g, 38 mmol, synthesized according to WO 2010106333A1 and WO 2012038743A1) and KOH (8 g, 142 mmol) in 1,4-dioxane (100 mL). The reaction mixture was stirred at 75°C for 4 hours and then cooled to room temperature. The solution was diluted with saturated _Na2S2O3 (aqueous _{solution )} , and the resulting precipitate was filtered and dried to obtain a yellow solid (4.1 g). The filtrate was allowed to stand for 3 days, and the resulting precipitate was filtered to produce an additional 3.55 g of product. The combined yield was 7.65 g, 72%. ¹ H NMR (400MHz, CDCl ₃ ) δppm7.65 (d, J＝6.0Hz, 1H), 8.13 (d, J＝6.0Hz, 1H), 14.22 (s, 1H). m/z (ESI) ⁺ :280[M+H] ⁺

Step 2

Synthesis of 1-(4-methoxybenzyl)-4-chloro-3-iodo-1H-pyrazolo[4,3-c]pyridine (Intermediate 2):

At room temperature, 4-methoxybenzyl chloride (0.5 mL, 3.6 mmol) was added to a mixture of intermediate 1 (1 g, 3.6 mmol) and KOH (0.3 g, 5.4 mmol) in DMF (10 mL). The resulting mixture was stirred at room temperature for 2.5 hours and then concentrated under reduced pressure. The residue was dissolved in EtOAc and washed with water, and the organic phase was dried and concentrated under reduced pressure. The residue was purified by flash chromatography using a 0 to 30% EtOAc/PE gradient to give a solid mixture of N1:N2 positional isomers (N1:N2=9:1, 1.3 g, 93%). Major N1 isomer: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.70 (s, 3H), 5.57 (s, 2H), 6.87 (d, J=8.8 Hz, 2H), 7.22 (d, J=8.4 Hz, 2H), 7.80 (d, J=4.8 Hz, 1H), 8.17 (d, J=4.4 Hz, 1H). m/z (ESI) ⁺ : 400 [M+H] ⁺ .

Step 3

Synthesis of 1-(4-methoxybenzyl)-3-iodo-N-methyl-1H-pyrazolo[4,3-c]pyridin-4-amine (Intermediate 3):

Methylamine (6 mL, 40% aqueous solution) was added to a solution of intermediate 2 (1.0 g, 2.50 mmol) in n-BuOH (6 mL). The mixture was heated to 170° C. in a sealed reactor with stirring for 5 hours, then concentrated under reduced pressure. The residue was purified by flash chromatography, eluting with 0-50% EtOAc/DCM to give a white solid (0.84 g, 85%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 1.71(m,2H),2.24(m,4H),4.43(s,2H),4.94(m,1H),6.80(d,J＝6.0Hz,1H),7.41(m,2H),7 .59(d,J＝6.8Hz,1H),7.78(d,J＝6.0Hz,1H),7.98(d,J＝7.2Hz,1H),13.28(s,1H); m/z(ESI) ⁺ :395[M+H] ⁺ .

Step 4

Synthesis of (E)-methyl 3-(4-(methylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridin-3-yl)acrylate (Intermediate 4):

To a mixture of intermediate 3 (595 mg, 1.51 mmol) and tetrabutylammonium iodide (1.11 g, 3.02 mmol) in DMF/water/triethylamine (26 mL/2.8 mL/2.8 mL) was added methyl acrylate (1.30 g, 15.08 mmol) and Pd(dppf) _Cl₂ (220 mg, 0.3 mmol) at room temperature under argon. The resulting mixture was heated at 70°C in a sealed reactor overnight and then concentrated under reduced pressure. The crude residue was dissolved in EtOAc and washed with saturated brine. The organic phase was dried over _Na₂SO₄ , filtered, _and concentrated under reduced pressure. The product was purified by flash chromatography with a gradient elution of 0 to 60% ethyl acetate/petroleum ether to give a brown gummy solid (80%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 2.93(d,J＝4.4Hz,3H),3.69(s,3H),3.75(s,3H),5.49(s,2H),6.72(m,2H),6.87(d,J＝8.4Hz,2H),6 .92(d,J＝6.0Hz,1H),7.22(d,J＝8.0HZ,2H),7.83(d,J＝6.0Hz,1H),8.11(d,J＝17.6Hz,1H); m/z(ESI) ⁺ :353[M+H] ⁺ .

Step 5

Synthesis of methyl 3-(4-(methylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridin-3-yl)propanoate (Intermediate 5):

10% Pd/C (0.1 g) and acetic acid (2 mL) were added to a solution of intermediate 4 (317 mg, 0.90 mmol) in ethyl acetate (10 ml) and methanol (10 mL). The resulting mixture was stirred at room temperature overnight under an _H atmosphere and then filtered through celite. The filter cake was washed twice with EtOAc and the combined filtrate was concentrated under reduced pressure to give a white solid (90%). The residue was used in the next reaction without further purification. m/z (ESI) ⁺ : 355 [M+H] ⁺ .

Step 6

Synthesis of 3-(4-(methylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridin-3-yl)-propionic acid (Intermediate 6):

At room temperature, NaOH (160 mg, 4.0 mmol) was added to a solution of intermediate 5 (283 mg, 0.80 mmol) in methanol (15 mL) and water (3 mL). The resulting mixture was stirred at 40° C. for 4 hours, then adjusted to pH=4 with acetic acid, concentrated under reduced pressure, and the residue was purified by flash chromatography using a gradient elution of 0 to 30% methanol/DCM to give a yellow solid (68%). ¹ H NMR (400MHz, DMSO-d ₆ )δppm:2.68(d,J＝7.2Hz,2H),2.91(d,J＝4.4Hz,3H),3.21(t,J＝7.2Hz,2H),3.70(d,J＝6.0Hz,3H),5.33(d,J＝8.4Hz,2H),6. 33(d,J＝6.8Hz,1H),6.74(d,J＝10.4Hz,1H),6.86(d,J＝10.0Hz,2H),7.13(d,J＝8.4Hz,2H),7.72(d,J＝10.0Hz,1H),12.20(br s,1H); m/z(ESI) ⁺ :341[M+H] ⁺ .

Step 7

Synthesis of 6-methyl-2-(4-methoxybenzyl)-2,6,8,9-tetrahydro-7H-1,2,5,6-tetraazabenzo[cd]azulene-7-one (Intermediate 7):

Under argon, HOBt (2.06 g, 15.21 mmol) and DIPEA (1.97 g, 15.21 mmol) were added to a solution of intermediate 6 (4.31 g, 12.67 mmol) in anhydrous THF (250 mL). The mixture was cooled to 0°C, and EDCI (2.92 g, 15.21 mmol) was added. After stirring at 0°C for 0.5 hours, the reaction mixture was slowly warmed to room temperature and stirred overnight, then quenched with water (100 mL). Most of the THF was removed by concentration under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was washed with saturated brine (100 mL), dried _{over Na2SO4} , and concentrated under reduced pressure. The residue was purified by flash chromatography (EtOAc/PE gradient elution) to give a white solid (75%). ¹ H NMR (400MHz, DMSO-d ₆ )δppm:2.94(m,2H),3.12(m,2H),3.52(s,3H),3.70(s,3H),5.50(s,2H),6.87(d,J＝8. 8Hz,2H),7.23(d,J＝8.4Hz,2H),7.43(d,J＝6.0Hz,1H),8.16(d,J＝6.0Hz,1H); m/z(ESI) ⁺ :323[M+H] ⁺

Step 8

Synthesis of 6-methyl-2,6,8,9-tetrahydro-7H-1,2,5,6-tetraazabenzo[cd]azulene-7-one (Example 1):

Intermediate 7 was dissolved in TFA (150 mg, 5 mL) and stirred overnight at 90 ° C in a sealed reactor. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was dissolved in a mixed solvent of DCM/MeOH (1:1, v/v, 10 mL), neutralized with K ₂ CO ₃ , filtered, washed, and concentrated under reduced pressure. The residue was then purified by flash chromatography using a 0 to 30% methanol/DCM gradient elution, followed by purification on a reverse phase C-18 column using a 5 to 60% MeCN/water gradient elution to obtain the product as a white solid (75%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 2.94(s,2H),3.13(s,2H),3.49(s,3H),7.18(s,1H),8.14(s,1H),13.30(br s,1H); m/z(ESI) ⁺ :203[M+H] ⁺ .

Examples 2-14

Examples 2-14 were prepared analogously to Example 1, substituting the appropriate amine for methylamine in Step 3.

Example 15

Example 15 was prepared analogously to Example 14, substituting methyl methacrylate for methyl acrylate in Step 4.

Example 16

Synthesis of 6-methyl-6,7,8,9-tetrahydro-2H-1,2,5,6-tetraazabenzo[cd]azulene (Example 16):

At 0 ° C, BH ₃ (1M in 5mL of THF) was added to a solution of intermediate 7 (328mg in 10mL of anhydrous THF, 1.02mmol) in Example 1. After stirring at room temperature for 16 hours under argon protection, methanol (10mL) was added to quench the reaction mixture and then concentrated under reduced pressure. The crude intermediate product 8 was dissolved in TFA (5mL) and stirred overnight at 90 ° C in a sealed reactor. After cooling to room temperature, the mixture was concentrated under reduced pressure and dissolved in DCM/MeOH (1: 1, v/v, 10mL), neutralized with K ₂ CO ₃ , filtered and concentrated under reduced pressure. The residue was then purified by flash chromatography, eluting with a 0 to 30% methanol/DCM gradient, and then purified by reverse phase C-18 column, eluting with a 5 to 60% MeCN/water gradient to give a yellow solid (two-step yield 15%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 2.05 (m, 2H), 2.98 (m, 2H), 3.33 (s, 3H), 3.66 (m, 2H), 6.74 (d, J = 6.0Hz, 1H), 7.70 (d, J = 6.0Hz, 1H), 13.07 (br s,1H); m/z(ESI) ⁺ :189[M+H] ⁺ .

Examples 17-29

Examples 17-29 were prepared analogously to Example 16, as shown below, substituting the appropriate intermediate for Intermediate 7, which was prepared in Step 3 (Example 1) by substituting the appropriate amine for methylamine.

Example 30

Example 30 was prepared analogously to Example 16, as shown below, substituting the appropriate intermediate for Intermediate 7, which was prepared by substituting the appropriate amine for methylamine in Step 3 (Example 1) and methyl methacrylate for methyl acrylate in Step 4 (Example 1).

Example 31

Step 1

Synthesis of methyl 2-(4-(methylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridin-3-yl)benzoate (Intermediate 9):

A mixture of Intermediate 3 (1.8 g, 4.56 mmol), (2-(methoxycarbonyl)phenyl)boronic acid (1.23 g, 6.85 mmol), and _{K₃PO₄ (} 1.94 g, 9.13 mmol) in toluene (50 mL) was deoxygenated with argon, followed by the addition of Pd(OAc) _₂ (103 mg, 0.46 mmol). The mixture was transferred to a sealed autoclave and heated to 95°C for 16 hours. After concentration, the residue was partitioned between EtOAc (300 mL) and water (150 mL). The organic layer was washed with water (100 mL x 2), dried over _{anhydrous Na₂SO₄} , filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography using a 0 to 65% EtOAc/PE gradient to afford a light yellow foamy solid (1.44 g, 78%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 2.80(d,J＝8.8Hz,3H),3.44(s,3H),3.70(s,3H),5.11(m,1H),5.46(s,2H),6.90(m,3H),7.18(d,J＝8.8Hz,2H),7.55( d,J＝7.6Hz,1H),7.62(t,J＝7.6Hz,1H),7.71(t,J＝7.6Hz,1H),7.81(d,J＝6.0Hz,1H),7.88(d,J＝7.6Hz,1H); m/z(ESI) ⁺ :403[M+H] ⁺ .

Step 2

Synthesis of 2-(4-(ethylamino)-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridin-3-yl)benzoic acid (Intermediate 10):

At room temperature, NaOH (700 mg, 17.78 mmol) was added to a solution of intermediate 9 (1.44 g, 3.55 mmol) in methanol (20 mL) and water (5 mL). The resulting mixture was stirred at 45 ° C for 4 hours, and the pH value of the mixture was adjusted to 4 with acetic acid. After concentration under reduced pressure, the residue was purified by flash chromatography using a gradient elution of 0 to 30% methanol/DCM to give a white solid (1.3 g, 93%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 3.00(d,J＝4.8Hz,3H),3.71(s,3H),5.62(s,2H),6.89(d,J＝8.4Hz,2H),7.22(d,J＝8.4Hz,3H ),7.38(d,J＝7.2Hz,1H),7.48(d,J＝7.2Hz,1H),7.69(m,3H),8.02(d,J＝6.8Hz,1H),13.20(br s,1H); m/z(ESI) ⁺ :389[M+H] ⁺ .

Step 3

Synthesis of 11-(4-methoxybenzyl)-4-methyl-4,11-dihydro-5H-3,4,10,11-tetraazadibenzo[cd,h]azulene-5-one (Intermediate 11):

Under argon, _Et3N (1.02 g, 10.05 mmol) was added to a mixture of intermediate 10 (1.3 g, 3.34 mmol) and HATU (1.9 g, 5.02 mmol) in anhydrous DCM (40 mL). After stirring at room temperature for 16 h, the reaction mixture was concentrated under reduced pressure and the residue was purified by flash chromatography using a 0 to 30% EtOAc/PE gradient to give a white solid (1.11 g, 90%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 3.64(s,3H),3.70(s,3H),5.61(s,2H),6.89(d,J＝8.4Hz,2H),7.29(d,J＝8.4Hz,2H),7.39(d,J＝6.0 Hz,1H),7.51(t,J＝7.6Hz,1H),7.67(t,J＝7.4Hz,1H),8.10(d,J＝6.0Hz,1H),8.31(m,2H); m/z(ESI) ⁺ :371[M+H] ⁺ .

Step 4

Synthesis of 4-methyl-4,11-dihydro-5H-3,4,10,11-tetraazadibenzo[cd,h]azulene-5-one (Example 31):

A solution of Intermediate 11 (50 mg, 0.135 mmol) in TFA (3 mL) was heated to 90°C for 6 hours, then concentrated under reduced pressure and purified by flash chromatography using a gradient of 0 to 30% methanol in DCM to afford a white solid (45%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 3.66 (s, 3H), 7.16 (d, J=6.0 Hz, 1H), 7.52 (t, J=5.6 Hz, 1H), 7.69 (t, J=5.2 Hz, 1H), 8.08 (d, J=6.4 Hz, 1H), 8.35 (m, 2H), 13.73 (s, 1H). m/z (ESI) ⁺ : 251 [M+H] ⁺ .

Examples 32-44

Examples 32-44 were prepared analogously to Example 31, as shown below, substituting the appropriate intermediate for Intermediate 3, which was prepared in Step 3 (Example 1) by substituting the appropriate amine for methylamine.

Example 45

Synthesis of 4-methyl-5,11-dihydro-4H-3,4,10,11-tetraazadibenzo[cd,h]azulene (Example 45):

0 ℃, under argon protection, BH ₃ (1.6M, in THF, 4mL) is added to the intermediate 11 (260mg, 0.70mmol) in anhydrous THF (15mL) solution in Example 31. The mixture is stirred at room temperature for 3 hours, then quenched and concentrated under reduced pressure by adding methanol (6mL). The crude product 12 is dissolved in TFA (5mL) and stirred overnight at 90 ℃ in a sealed reactor. After cooling to room temperature, the mixture is concentrated under reduced pressure, and the residue is dissolved in DCM/MeOH (1: 1, v/v, 10mL), neutralized with K ₂ CO ₃ , filtered and concentrated. The residue is purified by flash chromatography, eluting with a 0 to 35% methanol/DCM gradient, then purified by reverse phase C-18 column, eluting with a 5 to 70% MeCN/water gradient to obtain an off-white solid (two-step yield 36%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 3.33(s,3H),4.86(s,2H),7.06(s,1H),7.50(m,2H),7.62(d,J＝6.8Hz,1H),7.73(d,J＝6.4Hz,1H),8.02(d,J＝7.2Hz,1H),12.68(br s,1H); m/z(ESI) ⁺ :237[M+H] ⁺ .

Examples 46-58

Examples 46-58 were prepared similarly to Example 45 according to the following scheme.

Examples 59, 60

Examples 59, 60 were prepared analogously to Example 44 according to the following scheme, substituting the appropriate phenylboronic acid for 2-(methoxycarbonyl)phenylboronic acid in Step 1 (Example 31) and substituting tetrahydro-2H-pyran-4-amine for methylamine in Step 3 (Example 1).

Examples 61, 62

Examples 61 and 62 were prepared analogously to Example 58 according to the following scheme, substituting the appropriate intermediate for Intermediate 11 (Example 45) by substituting the appropriate phenylboronic acid for 2-(methoxycarbonyl)phenylboronic acid in Step 1 (Example 31) and substituting tetrahydro-2H-pyran-4-amine for methylamine in Step 3 (Example 1).

Example 65

Step 1

Synthesis of ethyl 4-iodo-1-trityl-1H-pyrazole-3-carboxylate (Intermediate 16):

A mixture of ethyl 4-iodo-1H-pyrazole-3 _- carboxylate (8.0 g, 30.0 mmol), _K₂CO₃ (12.42 g, 90.0 mmol), and TrtCl (10.06 g, 36.0 mmol) in MeCN (200 mL) was heated to 90°C for 15 hours. After cooling to room temperature, the mixture was filtered, and the filter cake was washed with ethyl acetate (100 mL). The combined filtrates were concentrated under reduced pressure, and the residue was purified by flash chromatography using a 0-15% EtOAc/PE gradient to afford a white crystalline solid (10.0 g, 67%). ¹ H NMR (400 MHz, DMSO- _d₆ ) δ ppm: 1.28 (t, J = 7.2 Hz, 3H), 4.27 (m, J = 7.2 Hz, 2H), 7.02 (m, 6H), 7.29–7.50 (m, 9H), 7.56 (s, 1H).

Step 2

Synthesis of ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-trityl-1H-pyrazole-3-carboxylate (Intermediate 17):

Under argon, a solution of Intermediate 16 (2.1 g, 4.13 mmol) and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.31 g, 12.40 mmol) in anhydrous THF (20 mL) was cooled to -75°C, and n-BuLi (7.75 mL, 12.40 mmol, 1.6 M in n-hexane) was added dropwise, maintaining the reaction temperature below -70°C. After the addition was complete, the mixture was stirred at -75°C for 1 hour. Water was added to quench the reaction mixture, and the mixture was partitioned between water and EtOAc. The organic layer was washed with saturated aqueous _NH4Cl (100 mL), water (100 mL), and saturated brine (100 _mL ), dried ( _Na2SO4 ), and concentrated under reduced pressure. The residue was purified by flash chromatography using a gradient of 0 to 30% EtOAc/PE to give a white solid (0.5 g, 24%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm: 1.23 (s, 12H), 1.28 (t, J=5.2 Hz, 3H), 4.24 (q, J=5.2 Hz, 2H), 7.04 (m, 6H), 7.39 (m, 9H), 7.47 (s, 1H).

Step 3

Synthesis of ethyl 4-(1-(4-methoxybenzyl)-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1-trityl-1H-pyrazole-3-carboxylate (Intermediate 18):

Under argon, a solution of Intermediate 3a (3.25 g, 7.0 mmol, prepared analogously to Intermediate 3 in Example 1 by replacing methylamine with tetrahydro-2H-pyran-4-amine in Step 3) and Intermediate 17 (3.92 g, 7.7 mmol), _Cs₂CO₃ (6.85 g, 21 mmol), and Pd(dppf) _{Cl₂ in} anhydrous DMF (20 mL) was sealed in a reaction vessel. The mixture was stirred at 90°C for 16 hours and cooled to room temperature, and 200 mL of saturated _NH₄Cl solution was added to the reaction mixture. After extraction with ethyl acetate (3 x 150 mL), the combined organic layers were washed with 150 mL of saturated _NH₄Cl solution, dried _{over Na₂SO₄} , and concentrated under reduced pressure. The residue was purified by flash chromatography using a gradient elution with 0% to 80% EtOAc/PE to yield a white solid (3.0 g, 60%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 0.67(t,J＝6.8Hz,3H),1.15(m,2H),1.80(m,2H),3.40(m,2H),3.72(m,5H),3.95(m,2H),4.06(m,1H) ,4.72(d,J＝7.6Hz,2H),5.46(s,2H),6.86(m,3H),7.16(m,8H),7.42(m,10H),7.75(m,2H).m/z(ESI) ⁺ :719[M+H] ⁺ .

Step 4

Synthesis of 4-(1-(4-methoxybenzyl)-4-(tetrahydro-2H-pyran-4-ylamino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1-trityl-1H-pyrazole-3-carboxylic acid (Intermediate 19):

A mixture of Intermediate 18 (3.0 g, 4.34 mmol) and NaOH (0.87 g, 21.71 mmol) in THF/ _H₂O (1:1, v/v, 16 mL) was stirred at 50°C for 60 hours and cooled to room temperature. The THF was removed from the mixture under reduced pressure, and the pH of the mixture was adjusted to 3 with HCl (1 N). The mixture was extracted with DCM (100 mL x 3), and the organic layers were combined, dried ( _Na₂SO₄ ), and concentrated to give a crude product (2.5 g), which was used directly in the next reaction without purification. _m /z (ESI) ^⁺ : 691 [M+H] ^⁺ .

Step 5

Synthesis of 5-(4-methoxybenzyl)-9-(tetrahydro-2H-pyran-4-yl)-2-trityl-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(2H)-one (Intermediate 20):

A mixture of intermediate 19 (2.5 g, 3.26 mmol), HATU (2.06 g, 5.43 mmol) and TEA in _CH2Cl2 (50 _mL ) was stirred at room temperature for 16 h. The mixture was then concentrated under reduced pressure and the residue was purified by flash chromatography eluting with a 0% to 80% EtOAc/PE gradient to give a white solid (3.0 g, 60%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 1.60(d,J＝10.4Hz,2H),2.80(m,2H),3.40(m,2H),3.69(s,3H),3.97(m,2H),5.51(s,2H),5.67(m,1 H),6.86(d,J＝8.8Hz,2H),7.18(m,8H),7.41(m,10H),7.75(s,1H),8.15(d,J＝6.0Hz,1H).m/z(ESI) ⁺ :673[M+H] ⁺ .

Step 6

Synthesis of 9-(tetrahydro-2H-pyran-4-yl)-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(2H)-one (Example 65):

A mixture of intermediate 20 (50 mg, 0.080 mmol) and TFA (3 mL) was stirred in a sealed reactor at 90° C. for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography on a reverse phase C-18 column eluting with a gradient of 5% to 60% acetonitrile in water to give a white solid (12 mg, 60%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 1.62(m,2H),2.84(m,2H),3.76(m,2H),3.98(m,2H),5.81(m,1H),7.15(d,J＝5.6H z,1H),8.05(s,1H),8.09(d,J＝5.6Hz,1H),13.41(s,1H),14.01(s,1H).m/z(ESI) ⁺ :311[M+H] ⁺ .．

Examples 66 and 67

Step 1

Synthesis of 5-(4-methoxybenzyl)-9-(tetrahydro-2H-pyran-4-yl)-2-trityl-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(2H)-one (Intermediate 21):

A solution _of Intermediate 20 (1.0 g, 1.49 mmol) from Example 65 in _CH₂Cl₂ /TFA (22 mL, 10:1, v/v) was stirred at room temperature for 3 hours. The reaction mixture was quenched with 50 mL _of saturated _Na₂CO₃ solution, and the _CH₂Cl₂ in the mixture was removed under reduced _pressure . The solid in the mixture was collected by filtration, and the filter cake was washed with water (20 mL x 3) to yield a white solid (0.42 g, 66%), which was used in the next reaction without further purification. m/z (ESI) ^⁺ : 431 [M+H] ^⁺ .

Step 2

Synthesis of 5-(4-methoxybenzyl)-2-methyl-9-(tetrahydro-2H-pyran-4-yl)-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(2H)-one (Intermediate 22) and 5-(4-methoxybenzyl)-1-methyl-9-(tetrahydro-2H-pyran-4-yl)-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(1H)-one (Intermediate 23):

In a sealed reaction vessel at room temperature, _CH₃I (145 mg, 1.02 mmol) was added to a mixture _of Intermediate 21 (220 mg, 0.51 mmol) and _Cs₂CO₃ (333 mg, 1.02 mmol) in anhydrous DMF (5 mL). After stirring at 60°C for 5 hours, the mixture was cooled to room temperature and poured into 20 mL of saturated aqueous _NH₄Cl solution, followed by extraction with EA (30 mL x 3). The combined organic layers were washed with saturated brine (20 mL x ₃ ), dried over anhydrous _Na₂SO₄ , and concentrated under reduced pressure to afford a crude mixture of Intermediates 22 and 23 (200 mg, white solid), which was used directly in the next reaction without further purification. m/z (ESI) ^⁺ : 445 [M+H] ^⁺ .

Step 3

Synthesis of 2-methyl-9-(tetrahydro-2H-pyran-4-yl)-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(2H)-one (Example 66) and 1-methyl-9-(tetrahydro-2H-pyran-4-yl)-5,9-dihydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene-10(1H)-one (Example 67):

A mixture of intermediates 22 and 23 (200 mg, 0.45 mmol) and TFA (5 mL) in a sealed reaction vessel was stirred for 16 h at 90° C. The mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography equipped with a reverse phase C-18 column eluting with a gradient of 5% to 50% acetonitrile/water to give 20 mg of the final product 66 and 42 mg of the final product 67.

Compound 66: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.63 (m, 2H), 2.80 (m, 2H), 3.46 (m, 2H), 3.94 (m, 2H), 4.13 (s, 3H), 5.71 (m, 1H), 7.10 (d, J=5.6 Hz, 1H), 7.97 (s, 1H), 8.06 (d, J=5.6 Hz, 1H), 13.41 (s, 1H). m/z (ESI) ⁺ : 325 [M+H] ⁺ .

Compound 67: ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.61 (m, 2H), 2.80 (m, 2H), 3.45 (m, 2H), 3.95 (m, 5H), 5.63 (m, 1H), 7.11 (d, J=5.6 Hz, 1H), 8.07 (d, J=5.6 Hz, 1H), 8.35 (s, 1H), 13.37 (s, 1H). m/z (ESI) ⁺ : 325 [M+H] ⁺ .

Example 68

Synthesis of pyrazole boron ester:

Step 1

Synthesis of ethyl 4-iodo-1H-pyrazole-3-carboxylate (25)

At room temperature, NIS (22.5 g, 100 mmol) was added to a solution of ethyl 1H-pyrazole-3-carboxylate (10.0 g, 71.4 mmol) in DCM (30 mL). The mixture was stirred for 24 hours. The reaction was then quenched with _H₂O (50 mL), and the mixture was extracted with EtOAc (60 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3) and dried over _Na₂SO₄ . After filtration, the filtrate was concentrated _under reduced pressure. The residue was purified by chromatography (PE:EtOAc = 1:5, v/v) to give the product, ethyl 4-iodo-1H-pyrazole-3-carboxylate (15.5 g, 82%). m/z (ESI) ⁺ : 267 [M+H] ⁺ .

Step 2

Synthesis of ethyl 4-iodo-1-isopropyl-1H-pyrazole-5-carboxylate (26) and ethyl 4-iodo-1-isopropyl-1H-pyrazole-3-carboxylate (27)

2-Iodopropane (12.9 g, 75.9 mmol) and _K₂CO₃ (16.0 g, 116 mmol) were added to a solution _of ethyl 4-iodo-1H-pyrazole-3-carboxylate (15.53 g, 58.4 mmol) in DMF (150 mL). The mixture was heated to 60°C and the reaction continued for 3 hours. The reaction was then quenched with water (300 mL) and extracted with EtOAc (200 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 3) and dried over _{Na₂SO₄ .} After filtration, the organic phase was concentrated under reduced pressure. The residue was purified by normal phase column chromatography (PE:EtOAc=10:1, v/v) to give ethyl 4-iodo-1-isopropyl-1H-pyrazole-5-carboxylate (Intermediate 26) (9.4 g, 52%), m/z (ESI) ⁺ : 309 [M+H] ⁺ , and eluted with PE:EtOAc=8:1 (v/v) to give ethyl 4-iodo-1-isopropyl-1H-pyrazole-3-carboxylate (Intermediate 27) (6.1 g, 33%), m/z (ESI) ⁺ : 309 [M+H] ⁺ .

Step 3

Synthesis of 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-5-carboxylic acid ethyl ester (28)

4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (3.56 g, 14.0 mmol), KOAc (2.7 g, 27.5 mmol), and Pd(dppf) _2Cl2 (300 mg) were added to a solution _of ethyl 4-iodo-1-isopropyl-1H-pyrazole-5-carboxylate (2.14 g, 6.95 mmol) in DMSO (20 mL). The mixture was heated to 100°C for 2 hours. The reaction was then quenched with _H2O (40 mL) and extracted with EtOAc (40 mL x 3). The combined organic phases were washed with saturated brine (40 mL x 3) and dried _{over Na2SO4} . After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (PE:EtOAc=10:1, v/v) to give ethyl 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-5-carboxylate (1.0 g, 93%). m/z (ESI) ⁺ : 309 [M+H] ⁺ .

Step 4

Synthesis of 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-3-carboxylic acid ethyl ester (29)

A solution of ethyl 4-iodo-1-isopropyl-1H-pyrazole-3-carboxylate (4.5 g, 14.6 mmol) and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (8.15 g, 43.8 mmol) in anhydrous THF (50 mL) was cooled to -75°C, and n-BuLi (17.5 mL, n-hexane solution, 1.6 N, 43.8 mmol) was added dropwise. The mixture was stirred at -75°C for 2 hours, and then quenched by the addition of 200 mL of saturated aqueous _NH4Cl solution. The mixture was extracted with ethyl acetate (100 mL x 3), and the combined organic layers were washed with 50 mL of saturated brine _, dried ( _Na2SO4 ), and concentrated under reduced pressure. The residue (yellow oil) was purified by flash chromatography using a 0 to 40% EtOAc/PE gradient to give a light yellow oil (2.0 g, 44%). ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 1.20-1.50 (m, 21H), 4.25 (q, J = 7.2Hz, 2H), 4.51-4.65 (m, 1H), 8.18 (s, 1H); m/z (ESI) ⁺ :309[M+H] ⁺ .

The following pyrazole boronate esters can be prepared analogously to 28 and 29 using iodomethane or iodoethane in step 2:

Synthesis Example 68:

Step 1

Synthesis of 3-iodo-1-(4-methoxybenzyl)-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-4-amine (30)

A mixture of 4-chloro-3-iodo-1-(4-methoxybenzyl)-1H-pyrazolo[4,3-c]pyridine (3.0 g, 7.5 mmol), 1-methylpiperidin-4-amine (2.57 g, 22.5 mmol), and DIEA (3.87 g, 30 mmol) in a 50 mL round-bottom flask was stirred overnight at 130°C. The reaction was then concentrated under reduced pressure, and the residue was purified by normal phase column chromatography using a DCM/MeOH gradient elution to afford 3-iodo-1-(4-methoxybenzyl)-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[4,3-c]pyridine-4-amine (2.7 g, 75%) as a brown solid. m/z (ESI) ⁺ : 478 [M+H] ⁺ .

Step 2

Synthesis of 1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1H-pyrazole-5-carboxylic acid ethyl ester (31)

Under _N2 protection, a solution of 3-iodo-1-(4-methoxybenzyl)-N-(1-methylpiperidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-4-amine (464 mg, 0.97 mmol), ethyl 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-5-carboxylate (300 mg, 0.97 mmol), Pd(dppf) _Cl2 (80 mg, 0.097 mmol), and _2NNa2CO3 (1 mL, 2 mmol) in 1,4- _dioxane (20 mL) was heated to 90°C and stirred overnight. The mixture was then diluted with EtOAc (100 mL), washed with water (30 mL x 2), saturated brine (30 mL) _, and dried over _Na2SO4 . The organic phase was filtered and the filtrate was concentrated to give a residue, which was purified by silica gel chromatography eluting with DCM/MeOH = 10/1 (v/v) to give ethyl 1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1H-pyrazole-5-carboxylate (300 mg, 58%) as a yellow oil. m/z (ESI) ⁺ : 532 [M+H] ⁺ .

Step 3

Synthesis of (1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1H-pyrazol-5-yl)methanol (32)

_LiAlH4 (182 mg, 4.79 mmol) was added portionwise to a solution of 1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1H-pyrazole-5-carboxylic acid ethyl ester (510 mg, 0.96 mmol) in anhydrous THF (20 mL) at 0°C. The mixture was stirred at room temperature for 1 hour. The reaction was then quenched with water (5 mL) and EtOAc (100 mL). The suspension was filtered, and the organic phase was separated and washed with saturated brine (15 mL), then dried _{over Na2SO4} . The organic phase was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with DCM/MeOH = 6/1 (v/v) to give (1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-1H-pyrazol-5-yl)methanol (300 mg, 64%) as a white solid. m/z (ESI) ⁺ : 490 [M+H] ⁺ .

Step 4

Synthesis of 1-isopropyl-5-(4-methoxybenzyl)-9-(1-methylpiperidin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene (33)

Methanesulfonyl chloride (144 mg, 1.22 mmol) was added dropwise to a solution of (1-isopropyl-4-(1-(4-methoxybenzyl)-4-((1-methylpiperidin-4-yl)amino)-1H-pyrazolo[ _4,3 -c]pyridin-3-yl)-1H-pyrazol-5-yl)methanol (300 mg, 0.61 mmol) and _Et3N (185 mg, 1.84 mmol) in THF (50 mL) at 0°C under N2. The reaction was then stirred at room temperature for 0.5 hours. TLC indicated the disappearance of the starting material, followed by the addition of t-BuOK (206 mg, 1.84 mmol). The reaction was heated to 60°C under _N2 protection and stirred for 1 hour. The reaction was then quenched with water (10 mL) and extracted with EtOAc (100 mL). The organic phase was washed with saturated brine (30 mL) and dried _{over Na2SO4} . The organic phase was filtered and the filtrate was concentrated to give a residue, which was purified by silica gel chromatography eluting with DCM/MeOH = 6/1 (v/v) to give 1-isopropyl-5-(4-methoxybenzyl)-9-(1-methylpiperidin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene (200 mg, 69%) as a colorless oil. m/z (ESI) ⁺ : 472 [M+H] ⁺ .

Step 5

Synthesis of 1-isopropyl-9-(1-methylpiperidin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene (Example 68)

A mixture of 1-isopropyl-5-(4-methoxybenzyl)-9-(1-methylpiperidin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene (200 mg, 0.42 mmol) in TFA (5 mL) was stirred at 70° C. overnight and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC, and the collected eluents were combined, adjusted to pH=8 with saturated aqueous NaHCO ₃ solution, extracted with EtOAc (10 mL×3), and filtered through Na ₂ SO The organic phase was _dried and filtered, and the filtrate was concentrated under reduced pressure to give 1-isopropyl-9-(1-methylpiperidin-4-yl)-1,5,9,10-tetrahydro-1,2,4,5,8,9-hexaazabenzo[cd]cyclopenta[h]azulene (110 mg, 74%) as a white solid. ¹ H NMR (400MHz, DMSO-d ₆ )δppm1.44(d,J＝6.0Hz,6H),1.63-1.71(m,2H),1.91-2.12(m,4H),2.22-2.32(m,3H),2.92(s,2H) ,4.47(s,2H),4.62(m,1H),4.93-4.96(m,1H),6.7,9(d,J＝6.0Hz,1H),7.78-7.81(m,2H),12.95(br s,1H); m/z(ESI) ⁺ :352[M+H] ⁺ .

Examples 69-88

Examples 69-88 were prepared analogously to Example 68 using the appropriate amine in Step 1 and the appropriate pyrazole boronic ester in Step 2. For Examples 69 and 76, the appropriate pyrazole boronic ester was Intermediate 17 in Step 2 of Example 65.

Example 89

Step 1

Synthesis of 4-iodo-6-(trifluoromethyl)nicotinic acid (34)

In a 3000 mL 3-necked round-bottom flask, under nitrogen protection, a solution of 2,2,6,6-tetramethylpiperidine (34.5 g, 141 mmol) in anhydrous THF (600 mL) was cooled to -78°C and maintained at -78°C. n-BuLi (98 mL, 2.4 M hexane solution) was added and stirred for 0.5 hour. A solution of 6-trifluoromethylnicotinic acid (15 g, 78 mmol) in anhydrous THF (50 mL) was then added dropwise, and the mixture was stirred at -78°C for 2.5 hours. Subsequently, a solution of _I2 (29.3 g, 117 mmol) in anhydrous THF (50 mL) was slowly added, and the reaction was also stirred at -78°C for 1 hour. The reaction was then quenched with 100 mL of saturated aqueous NH ₄ Cl solution. The pH of the reaction solution was adjusted to 4-5 with 1N HCl, and the mixture was extracted with DCM (400 mL × 3), dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified on a normal phase silica gel column (PE/EA/AcOH, 1:10:0.01, v/v/v) to afford Compound 34 (10 g, 40%) as a yellow solid. m/z (ESI) ⁻ : 316 [MH] ⁻ .

Step 2

Synthesis of 4-iodo-6-(trifluoromethyl)nicotinate (35)

A mixture of intermediate 34 (10 g, 31 mmol), K ₂ CO ₃ (8.7 g, 63 mmol), and MeI (5.37 g, 37.9 mmol) in DMF (25 mL) was stirred overnight at room temperature. H ₂ O (300 mL) was then added to the reaction mixture. The mixture was filtered and the filter cake was washed with water (20 mL) and air-dried to afford compound 35 (10 g, crude) as a yellow solid. m/z (ESI) ⁺ : 332 [M+H] ⁺ .

Step 3

Synthesis of methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6-(trifluoromethyl)nicotinate (36)

Under _N₂ protection, Intermediate 35 (10.0 g, 30.0 mmol), bis(pinacolato)diboron (11.5 g, 45.3 mmol), KOAc (5.9 g, 60.4 mmol), and Pd(dppf) _₂Cl₂ (3.31 g, 4.53 mmol) were mixed in 1,4- _dioxane (100 mL), heated to 95°C, and maintained at this temperature with stirring for 18 hours. The mixture was then cooled and concentrated, and the residue was partitioned between EtOAc (300 mL) and water (80 mL). The organic layer was washed with water (200 mL x 2), dried over _{anhydrous Na₂SO₄} , filtered, and concentrated under reduced pressure. The residue was purified by normal phase silica gel column chromatography (PE/EA, v/v, 20:1 to 10:1 gradient) to give the product (2.2 g) as a yellow solid. m/z (ESI) ^⁺ : 250 [M+H] ^⁺ .

Step 4

Synthesis of 4-(1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-6-(trifluoromethyl)nicotinic acid (37)

A solution of compound _3a (1.23 g, 2.66 mmol), intermediate 36 (2.2 g, 6.6 mmol) and K ₂ CO ₃ (733 mg, 5.32 mmol) in 1,4-dioxane/H ₂ O (4:1, v/v, 20 mL) was degassed with N 2 . Subsequently, PPh ₃ (175 mg, 0.67 mmol) and Pd(PPh ₃ ) ₄ (185 mg) were added to the above solution. Under nitrogen protection, the mixture was stirred at 100 ° C for 18 hours. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by normal phase silica gel column chromatography (DCM/MeOH, v/v, 5:1 to 2:1 gradient elution) to obtain compound 37 as a yellow solid (1.0 g, 69%). m/z (ESI) ⁺ : 528 [M+H] ⁺ .

Step 5

Synthesis of (4-(1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)-6-(trifluoromethyl)pyridin-3-yl)methanol (38)

A mixture of intermediate 37 (1 g, 1.9 mmol), _AlCl₃ (234 mg, 1.9 mmol), and _LiAlH₄ (261 mg, 7.86 mmol) in anhydrous THF (40 mL) was stirred at 0°C for 1 hour. _H₂O (0.26 mL), 15% NaOH (aqueous solution, 0.26 mL), and _H₂O (0.8 mL) were added sequentially. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel column chromatography (PE/EA, v/v, 5:1 to 2:1 gradient elution) to give the product (240 mg, 24.6%) as a yellow oil. m/z (ESI) ^⁺ : 514 [M+H] ^⁺ .

Step 6

Synthesis of 11-(4-methoxybenzyl)-4-(tetrahydro-2H-pyran-4-yl)-8-(trifluoromethyl)-5,11-dihydro-4H-3,4,7,10,11-pentaazadibenzo[cd,h]azulene (39)

At 0°C, MsCl (112 mg, 0.97 mmol) was added dropwise to a solution of intermediate 38 (250 mg, 0.49 mmol) and TEA (198 mg, 1.95 mmol) in anhydrous THF (20 mL). After stirring at 0°C for 0.5 hours, t-BuOK (219 mg, 1.95 mmol) was added. The reaction was then stirred at 55°C for 1.5 hours and concentrated under reduced pressure to give a residue. The residue was purified by normal phase silica gel column chromatography (PE/EA, 1:1, v/v) to give compound 39 (230 mg, 95%) as a yellow solid. m/z (ESI) ⁺ : 496 [M+H] ⁺ .

Step 7

Synthesis of 4-(tetrahydro-2H-pyran-4-yl)-8-(trifluoromethyl)-5,11-dihydro-4H-3,4,7,10,11-pentaazadibenzo[cd,h]azulene (Example 89)

Compound 39 (230 mg, 0.46 mmol) was dissolved in TFA (3 mL) in a microwave reaction tube and then microwave-assisted heated to 105°C with stirring for 1 hour. The reaction was then concentrated under reduced pressure and the residue was purified by preparative HPLC to give a white solid (55 mg, 33.1%). ¹ H-NMR (400MHz, DMSO-d ₆ ) δppm 1.76(d,J＝10.6Hz,2H),2.18(dt,J＝11.5,7.7Hz,2H),3.47(t,J＝11.4Hz,2H),4.04(dd,J＝11.1,3.6Hz,2H),4.41(t ,J＝10.2Hz,1H),4.89(s,2H),7.24(d,J＝6.8Hz,1H),7.86(d,J＝6.9Hz,1H),8.27(s,1H),9.19(s,1H),14.70(s,1H); ¹⁹ F-NMR (376MHz, DMSO-d ₆ ) δppm-66.72,-74.41; m/z (ESI) ⁺ :376[M+H] ⁺ .

Examples 90-92

Examples 90-92 were prepared analogously to Example 89 using the appropriate pyridine in step 1.

Example 93

Step 1

Synthesis of 2-((1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)ethanol (40)

A mixture of 3-iodo-1-(4-methoxybenzyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[4,3-c]pyridin-4-amine (1.5 g, 3.23 mmol), 2-(methylamino)ethanol (1.98 g, 32.3 mmol), cuprous iodide (123 mg, 0.65 mmol), L-proline (150 mg, 1.3 mmol) and t-BuOK (621 mg, 6.46 mmol) in DMSO (15 mL) was stirred at 100 °C overnight. The mixture was then diluted with EtOAc (100 mL), washed with water (30 mL x 2) and saturated brine (30 mL x 1), and dried over Na ₂ SO _{4 .} The mixture was filtered and the filtrate was concentrated. The residue was purified by normal phase silica gel column chromatography eluting with PE/EtOAc = 2/1 (v/v) to give 2-((1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)ethanol (760 mg, 57%) as a colorless oil. m/z (ESI) ⁺ : 412 [M+H] ⁺ .

Step 2

Synthesis of ethyl 2-((1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)methanesulfonate (41)

Methanesulfonyl chloride (429 mg, 3.7 mmol) was added dropwise to a solution of 2-((1-(4- _{methoxybenzyl} )-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)ethanol (760 mg, 1.85 mmol) and _Et3N (560 mg, 5.55 mmol) in THF (76 mL) at 0°C under N2 protection, and the mixture was stirred at room temperature for 0.5 h. The reaction was quenched with water (10 mL) and extracted with EtOAc (20 mL × 2). The combined organic phases were washed with saturated brine (20 mL × 1) and dried over Na ₂ SO _4. The organic phase was filtered and the filtrate was concentrated. The residue was purified by normal phase silica gel column chromatography with PE/EtOAc = 3:1 (v/v) as the eluent to give ethyl 2-((1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)methanesulfonate (489 mg, 54%) as a colorless oil. m/z (ESI) ⁺ : 490 [M+H] ⁺ .

Step 3

Synthesis of 2-(4-methoxybenzyl)-9-methyl-6-(tetrahydro-2H-pyran-4-yl)-6,7,8,9-tetrahydro-2H-1,2,5,6,9-pentaazabenzo[cd]azulene (42)

t-BuOK (312 mg, 2.79 mmol) was added to a solution of ethyl 2-((1-(4-methoxybenzyl)-4-((tetrahydro-2H-pyran-4-yl)amino)-1H-pyrazolo[4,3-c]pyridin-3-yl)(methyl)amino)methanesulfonate (455 mg, 0.93 mmol) in anhydrous THF (50 mL). The reaction was heated to 60° C. under N ₂ for 1 hour. The reaction was then quenched with water (10 mL) and extracted with EtOAc (100 mL). The organic phase was washed with saturated brine (30 mL) and dried over Na ₂ SO _4. The organic phase was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by normal phase silica gel column chromatography with PE/EtOAc=1/1 (v/v) as eluent to give 2-(4-methoxybenzyl)-9-methyl-6-(tetrahydro-2H-pyran-4-yl)-6,7,8,9-tetrahydro-2H-1,2,5,6,9-pentaazabenzo[cd]azulene (167 mg, 46%) as an oil. ¹ H-NMR (400MHz, CDCl ₃ ) δppm 1.57-1.66(m,2H),1.79(m,2H),3.06(s,3H),3.37(m,2H),3.60(m,4H),3.77(s,3H),4.04(m,2H),5.24(m ,3H),6.37(d,J＝6.0Hz,1H),6.82(d,J＝8.0Hz,2H),7.16(d,J＝8.0Hz,2H),7.78(d,J＝6.0Hz,1H); m/z(ESI) ⁺ :394[M+H] ⁺ .

Step 4

Synthesis of 9-methyl-6-(tetrahydro-2H-pyran-4-yl)-6,7,8,9-tetrahydro-2H-1,2,5,6,9-pentaazabenzo[cd]azulene (Example 93)

Under _N2 , a solution of 2-(4-methoxybenzyl)-9-methyl-6-(tetrahydro-2H-pyran-4-yl)-6,7,8,9-tetrahydro-2H-1,2,5,6,9-pentaazabenzo[cd]azulene (150 mg, 0.38 mmol) in TFA (7.5 mL) was heated to 70 °C for 8 hours, and then concentrated under reduced pressure to remove TFA. The residue was dissolved in EtOAc (150 mL), washed with _aqueous NaHCO3 (20 mL × 2) and saturated brine (20 mL × 1), and filtered through _Na2SO4 . The residue was purified by preparative TLC (DCM/MeOH=11/1, v/v) to give ₉ -methyl-6-(tetrahydro-2H-pyran-4-yl)-6,7,8,9-tetrahydro-2H-1,2,5,6,9-pentaazabenzo[cd]azulene (75 mg, 72%) as a white solid. ¹ H-NMR (400MHz, DMSO-d ₆ ) δppm 1.71(m,2H),1.87(m,2H),3.02(s,3H),3.44(m,4H),3.78(m,2H),3.98(m,2H),4.33(m,1H),6.84(s,1H),7.55(s,1H),12.35(br s,1H),12.98(br s,1H); ¹⁹ F-NMR (376MHz, DMSO-d ₆ ) δppm-74.41; m/z (ESI) ⁺ :274[M+H] ⁺ .

Example 94

Example 94 was prepared analogously to Example 93 using 2-(ethylamino)ethanol instead of 2-(methylamino)ethanol in Step 1.

The examples were assayed for LRRK2 inhibition and selectivity over other kinases such as JAK2 using the following method.

Materials and methods

Material

In the Adapta ^™ kinase assay (LRRK2 IC ₅₀ assay): the kinase reaction buffer contained 5× kinase buffer S (Life Technologies, PV5213) and 2 mM DTT (Life Technologies, P2325). The kinase LRRK2 G2019S protein (PV4881) and ERM (LRRKtide, PV5093) were from Life Technologies. The Adapta ^™ Universal Kinase Assay Kit (Life Technologies, PV5099) contained the following components: Adapta ^™ Eu-anti-ADP antibody (PV5097; 4 μg); 10 μM 647 ADP tracer (PV5098; 200 pmol); TR-FRET dilution buffer (PV3574; 100 ml); kinase quenching buffer (P2825; 1 ml); 10 mM ATP (PV3227; 500 μl); and 10 mM ADP (PV5096; 500 μl). LRRK2-IN-1 (1234480-84-2, HY-10875) was obtained from MedChem Express.

In the kinase assay ( _{JAK2_IC50} selectivity assay): JAK2 (Invitrogen, catalog number PV4288), ATP (Sigma, catalog number A7699-1g), DMSO (Sigma, catalog number D2650), DTT (Sigma, catalog number 43815), LANCE Ultra ULight ^™ -JAK-1 peptide (Perkin Elmer, catalog number TRF0121), LANCE Eu-W1024 anti-phosphotyrosine (PT66) (Perkin Elmer, catalog number AD0069), LANCE™ detection buffer (Perkin Elmer, catalog number CR97-100), Tofacitinib (PharmaBlock Sciences (Nanjing), Inc, catalog number PBN2011586-01); Equipment: Envision (Perkin Elmer), Bravo (Agilent); Consumables: 384-well intermediate plate (Greiner, catalog number 781280), 384-well assay plate (Perkin Elmer, catalog number 6007299),

In the Drosophila model: Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)] (ab133450) was from Abcam. ddc-GAL4 was from the Department of Medicine, Soochow University.

Adapta ^™ Kinase Assay

The Universal Kinase Assay is a uniform, fluorescence-based immunoassay for the detection of ADP. In contrast to ATP depletion assays, the assay is very sensitive to ADP formation, with the majority of the signal occurring within the initial 10-20% conversion of ATP to ADP. This makes the Universal Kinase Assay well-suited for use with low-activity kinases.

All assays were performed at room temperature (~21°C) and were linear with time and enzyme concentration under the conditions used. A 1× kinase reaction buffer solution was prepared from a 5× kinase buffer S stock solution (listed above). 10 ml of 1× kinase reaction buffer was prepared by adding 2 ml of the 5× stock solution to 8 ml of H ₂ O. 20 μl of 1 M DTT was added to this 1× kinase reaction buffer.

Kinase reactions were performed in a 10 μl volume in a low-volume 384-well plate. Typically, Greiner plates (Catalog #3674#, low volume, white wall, 784075) were used. Other untreated assay plates, which have not been tested, may also be suitable. The substrate concentration in this assay was 100 μM, and the 1× kinase reaction buffer consisted of 50 mM Tris-HCl pH 8.5, ₁₀ mM MgCl₂, 1 mM EGTA, 0.01% Brij-35, and 2 mM DTT, as well as any other additives that may be necessary for the specific kinase. The kinase reaction was allowed to proceed at room temperature for 1 hour, followed by the addition of 5 μl of a preparation of kinase quenching buffer (30 mM EDTA), Eu-labeled antibody (6 nM), and tracer (18.9 nM) in TR-FRET dilution buffer. The final concentrations of the antibody in the assay wells were 2 nM, the tracer was 6.3 nM, and the EDTA was 10 mM. The plate was allowed to equilibrate at room temperature for at least 30 minutes before being read on an Adapta ^™ TR-FRET plate reader configured for use.

The data presented in this document were generated using a BMG LABTECH PHERAstar plate reader using the appropriate filters and instrument settings for Adapta ^™ . Test compounds were screened in wells with 1% DMSO (final). For an 8-point titration, 5 serial dilutions were performed from the starting concentration.

Kinase Selectivity Assay

The assay involves two steps: an enzymatic step and a detection step using HTRF reagents. Step 1: During the enzymatic step, the substrate biotin is incubated with the kinase of interest. ATP is added to initiate the reaction. Step 2: The detection reagent captures the phosphorylated substrate, and the resulting TR-FRET is proportional to the level of phosphorylation.

Compound preparation: Dissolve the test compound in 30 mM DMSO and store this solution long-term at room temperature under a nitrogen hood. Dilute the 30 mM compound in DMSO by 3-fold, for a total of 11 concentrations. Aspirate 1 μl of the compound and subsequently dilute it 25-fold with kinase buffer. Mix thoroughly and equilibrate at room temperature for 30 minutes.

Kinase reaction: Transfer 2.5 μl of compound in kinase buffer (4x) to the assay plate using an Agilent Bravo. Rotate the plate; transfer 5 μl of enzyme mix to the assay plate using an Eppendorf electronic multichannel pipette. Rotate the plate; incubate the assay plate at room temperature (23°C) for 20 minutes; add 2.5 μl of kinase buffer containing ATP to the assay plate using a Multidrop. Rotate and seal the plate; incubate the assay plate at room temperature (23°C) for 90 minutes.

Stop the reaction: Use an Eppendorf electronic multichannel pipette to transfer 10 μl of detection reagent (2 nM LANCE Eu-W1024 anti-phosphotyrosine) to the assay plate. Vortex and seal the plate; incubate the assay plate at room temperature (23° C.) for 60 minutes.

Detection and reading: Excitation wavelength is 340nm, primary emission wavelength is 615nm, and secondary emission wavelength is 665nm (for Cryptate and Ulight, respectively). Read the plate with EnVision and obtain readings at two wavelengths; calculate the ratio of 665nm/615nm.

_IC50 estimates were obtained using XLfit (IDBS Inc.) software.

Drosophila model screening method

The Drosophila model was used to evaluate the examples in vivo. The GAL4/UAS system developed by Andrea Brand and Norbert Perrimon in 1993 [45] was used to generate transgenic flies expressing LRRK2-G2019S in dopamine (DA) neurons. The system has two parts: the GAL4 gene encoding the yeast transcriptional activator protein GAL4 and the enhancer UAS (upstream activating sequence) to which GAL4 specifically binds to activate gene transcription. The system utilizes the yeast GAL4 transcription factor that specifically binds to UAS. The UAS-wild-type-LRRK2 and UAS-G2019S-LRRK2 transgenes were combined with the dopa decarboxylase (ddc)-GAL4 driver [46] to express LRRK2-G2019S in DA neurons. GW5074 at 10 μM was used as a positive control. The negative control was a DMSO control (all compounds were dissolved in DMSO at a dilution of 1:1000). The flies were maintained at 25°C in a 12-h light and 12-h dark cycle. GW5074 was used as a positive control [47].

Survival rate

Twenty newly eclosed female fruit flies were collected and placed in food vials. Flies were transferred to fresh food vials every other day, at which time mortality was recorded.

Based on the Drosophila survival curve, the 50% survival time parameter represents the time at which half of the flies survive and is used to compare survival rates between different groups. The mean 50% survival time and standard error were calculated based on four independent experiments per group. Data were analyzed using GraphPad 6.0 software.

Climbing Assay

Negative geotaxis assay was used to analyze the locomotor ability of Drosophila. Twenty flies per vial (four vials per group) were subjected to a climbing test once a week.

The fruit flies to be tested were transferred to a vertical plastic tube (15 cm high, 1.5 cm in diameter). After standing at room temperature for 30 minutes, the fruit flies were gently knocked to the bottom of the tube, and the number of fruit flies that could climb to or above the test line within 10 seconds was counted and the percentage was calculated.

The climbing ability was analyzed based on three independent experiments for each vial, and the data were analyzed by GraphPad 6.0 software.

Kinase assay at week 6

Adult Drosophila heads were homogenized on ice, and kinase reactions were performed on brain lysates in kinase reaction buffer with ATP and DTT.

The lysates were then electrophoresed through a 12% SDS-PAGE gel and transferred to a PVDF membrane (Millipore). The membranes were blocked in TBST with 5% skim milk for 1 hour at room temperature and then incubated overnight at 4°C in anti-LRRK2pSer935 antibody (Abcam, ab133450) and anti-Flag antibody.

Proteins were detected using HRP-conjugated secondary antibodies and ECL detection reagents. The optical density of immunoblots was analyzed by Image J software, and the ratio of phosphorylated LRRK2 protein to Flag protein was calculated, and the data were analyzed by GraphPad 6.0 software.

Kinase Selectivity Assay

1. MKK1 assay

This is a two-step assay in which inactive MAPK (0.06 mg/ml) is activated by MKK1 (diluted in 25 mM Tris, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 0.01% Brij35, 1 mg/ml BSA) in 25.5 μl of 25 mM Tris, 0.1 mM EGTA, 0.01% Brij35, 10 mM magnesium acetate and 0.005 mM ATP. After incubation at room temperature for 30 minutes, 5 μl of the first reaction was pipetted into a second reaction mixture containing (final concentration) 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na 3 VO 4 , 0.66 mg/ml myelin basic protein (MBP), 10 mM magnesium acetate, and 0.05 mM [ 33 P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

2.MAPK2/ERK2 test.

MAPK/ERK2 targeting MBP (5–20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was measured in a final volume of 25.5 μl of 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected into a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

3.JNK1a1/SAPK1c experiment.

JNK1a1/SAPK1c (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) against ATF2 (activating transcription factor) was measured in a final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

4.SAPK 2a/p38 test.

SAPK 2a/p38 against MBP (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

5.SAPK 2b/p38β2 test.

SAPK2b/p38β2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed for MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

6.SAPK 3/p38g test.

SAPK 3/p38g against MBP (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

7.SAPK test.

SAPK 4/p38d (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed for MBP in a final volume of 25.5 μl containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 0.1% b-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

8.MAPKAP-K1a assay.

MAPKAP-K1a against KKLNRTLSVA (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed in a final volume of 25.5 μl containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 40 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

9.MAPKAP-K2 assay.

MAPKAP-K2 against KKLNRTLSVA (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed in a final volume of 25.5 μl containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

10.MSK1 trial.

MSK1 (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the modified crosstide peptide GRPRTSSFAEGKK was assayed in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

11.PRAK test.

PRAK (5-20 mU diluted in 50 mM b-sodium glycerophosphate pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against KKLRRTLSVA was assayed in a final volume of 25.5 μl containing 50 mM b-sodium glycerophosphate pH 7.5, 0.1 mM EGTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

12.PKA test.

PKA against kemptide (LRRASLG) (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was measured in a final volume of 25.5 μl containing 8 mM MOPS pH 7.5, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

13.PKCa test.

PKCa (5-20 mU, diluted in 20 mM Hepes pH 7.4, 0.03% Triton X-100) was measured against histone H1 in the presence of phosphatidylserine (PtdSerine) and DAG (0.1 mg/ml and 10 μg/ml) and 0.1 mM CaCl2. The assay was performed in a final volume of 25.5 μl containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1 mg/ml histone H1, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection into a P81 Unifilter plate with a 50 mM orthophosphoric acid wash buffer.

Preparation of PtdSer/DAG: 10 mg/ml PtdSer stock in MeOH/chloroform (1:2). Dry the required amount. Resuspend in an appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly sonicate (2 x 10-15 seconds, 10-15 second intervals). 10 mg/ml DAG stock in MeOH/chloroform (1:2). Dry the required amount. Add the sonicated PtdSer solution. Vortex and sonicate.

14.PDK1 trial.

PDK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 1 mg/ml BSA) was assayed for PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 100 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

15.ΔPH-PKBa-S473D assay.

ΔPH-PKBa-S473D (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against the modified crosstide peptide GRPRTSSFAEGKK was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

16.SGK test.

SGK (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against the modified crosstide peptide GRPRTSSFAEGKK was assayed in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

17.S6K1/P70S6K test.

S6K1/P70S6K (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1 mM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) was assayed against the substrate peptide (KKRNRTLTV) in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.1 mM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

18.GSK3b trial.

GSK3b (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed for phosphorylated GS2 peptide (YRRAAVPPSPSLSRHSSPHQS(PO4)EDEEE) in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 μM phosphorylated GS2 peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

19.ROCK-II (ROKa) test.

ROCK-II (ROKa) (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against the Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μM Long S6 substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

20.AMPK assay.

AMPK (5-20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35) was measured against the SAMS substrate peptide (HMRSAMSGLHLVKRR) in a final volume of 25.5 μl containing 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 0.4 mM SAMS peptide, 0.196 mM AMP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

21.CHK1 test.

In a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μM CHKtide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), CHK1 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% b-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA) was assayed against the CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

22.CK2 test.

CK2 (5-20 mU diluted in 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton-X100, CKII peptide (0.165 mM), 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed against CKII peptide (RRRDDDSDDD) in a final volume of 25.5 μl containing 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EGTA, 0.1% Triton X-100, 5 mM DTT, 50% glycerol and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

23.PBK test.

PBK (5-20 mU, diluted in 50 mM sodium b-glycerophosphate pH 7.0, 0.1% b-mercaptoethanol) against the phosphorylated b-peptide (KRKQISVRGL) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 8.6, 50 mM sodium b-glycerophosphate, 0.04 mM CaCl2, phosphorylated b-peptide (0.196 mM), 10 mM magnesium acetate, 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 15 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

24.LCK trial.

LCK (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against Cdc2 peptide (KVEKIGEGTYGVVYK) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3Vo4, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 15 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

25.CSK test.

CSK (5-20 mU, diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) against Cdc2 peptide (KVEKIGEGTYGVVYK) was assayed in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

26.CDK2/cyclin A assay.

CDK2/cyclin A (5-20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl) against histone H1 was measured in a final volume of 25.5 μl containing 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCl, histone H1 (1 mg/ml), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

27.DYRK 1A trial.

DYRK1A (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) against Woodtide was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

28.CK1 test.

CK1 (5-20 mU diluted in 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% Triton-X100, CKI peptide (0.5 mM), 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) was assayed against CKI peptide (RRKDLHDDEEDEAMSITA) in a final volume of 25.5 μl containing 20 mM Hepes pH 7.5, 0.15 M NaCl, 0.1 mM EGTA, 0.1% Triton X-100, 5 mM DTT, 50% glycerol and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

29.NEK6 trial.

NEK6 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the NEK6 peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% β-mercaptoethanol, NEK6 peptide (0.3 mM), 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

30.NEK2a trial.

5-20 mU of NEK2a (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against NEK2a peptide (RFRRSRRMI) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% β-mercaptoethanol, 300 μM NEK2a peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

31.MAPKAP-K1b/RSK2 assay.

MAPKAP-K1b (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed against the substrate peptide (KKLNRTLSVA) in a final volume of 25.5 μl containing 50 mM sodium b-glycerophosphate (pH 7.5), 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

32.IKKb test.

5-20 mU of IKKb (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (LDDRHDSGLDSMKDEEY) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

33.smMLCK test

5-20 mU of smMLCK (diluted in 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% b-mercaptoethanol) was assayed against the substrate peptide (KKRPQRATSNVFA) in a final volume of 25.5 μl containing 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 5 mM CaCl2, 10 μM calmodulin, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

34.PRK2 Trial

5-20 mU of PRK2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the Long S6 peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 30 μM Long S6 peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

35. MNK2ɑ assay

5-20 mU of MNK2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (eIF4E) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.5 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a 50 mM orthophosphoric acid wash buffer.

36.CAMK-1 assay

In the 25.5 μ l final volume containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl , 0.3 μ M calmodulin, 0.1% β-mercaptoethanol, 300 μ M substrate peptide, 10 mM magnesium acetate and 0.05 mM [ 33 P-g-ATP] (500-1000 cpm/pmole), 5-20 mU of CAMK-1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) directed against substrate peptide (YLRRRLSDSNF) is measured and incubated at room temperature for 30 minutes. The assay is stopped by adding 5 μ l of 0.5 M (3%) orthophosphoric acid. The assay solution is collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

37.PIM2 test

5-20 mU of PIM2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (RSRHSSYPAGT) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.5 mM CaCl2, 0.3 μM calmodulin, 0.1% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay solution was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

38.NEK7 trial

NEK7 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.01% Brij, 0.1% β-mercaptoethanol, the substrate peptide (0.3 mM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

39. JNK3ɑ1 assay

JNK3α1 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) against ATF2 (activating transcription factor) was measured in a final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

40. MAPKAP-K3 assay

5-20 mU of MAPKAP-K3 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) were assayed against the substrate peptide (KKLNRTLSVA) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, and 0.1% β-mercaptoethanol and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

41.ERK8 assay

5-20 mU of ERK8 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

42.MNK1 assay

5-20 mU of MNK1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (eIF4E) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.5 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a 50 mM orthophosphoric acid wash buffer.

43.SRPK1 assay

5-20 mU of SRPK1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against the substrate peptide (RSRSRSRSRSRSR) in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a 50 mM orthophosphoric acid wash buffer.

44.ΔPH-PKBβ(S474D) assay

ΔPH-PKBβ-S474D (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was assayed against a modified Crosstide peptide (GRPRTSSFAEGKK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

45.Aurora B trial

Aurora B (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was assayed against the substrate peptide (LRRLSLGLRRLSLGLRRLSLGLRRLSLG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

46.CHK2 test

CHK2 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1% b-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA) was assayed against the CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μl containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μM CHKtide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

47.Src test

Src (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed for the substrate peptide (KVEKIGEGTYGVVYK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

48.EF2K test

EF2K (5-20 mU, diluted in 50 mM Hepes pH 6.6, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was measured against the substrate peptide (RKKFGESKTKTKEFL) in a final volume of 25.5 μl containing 50 mM Hepes pH 6.6, 0.2 mM CaCl2, 0.3 μM calmodulin, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

49. MARK3 trial

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole), MARK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against CHKtide substrate (KKKVSRSGLYRSPSMPENLNRPR) was assayed and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

50.MST2 trial

MST2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 100 μM vanadate) was measured for MBP in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

51.PKD1 test

PKD1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed for substrate peptide (KKLNRTLSVA) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

52.PLK1 assay

PLK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadate) was assayed against the substrate peptide (ISDELMDATFADQEAKKK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

53.DYRK2 trial

DYRK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) against Woodtide (KKISGRLSPIMTEQ) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

54.JNK2 assay

JNK21 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) against ATF2 (activating transcription factor) was measured in a final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

55.DYRK3 trial

DYRK3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA) against Woodtide (KKISGRLSPIMTEQ) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

56.HIPK2 trial

5-20 mU of HIPK2 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

57.HIPK3 trial

5-20 mU of HIPK3 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

58.PAK4 trial

PAK4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed against the substrate peptide (RRRLSFAEPG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

59. PAK5 (PAK7) trial

PAK5 (PAK7) was assayed for substrate peptide (RRRLSFAEPG) (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

60. PAK6 trial

PAK6 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol, 1 mg/ml BSA) was assayed against the substrate peptide (RRRLSFAEPG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

61.CAMKKa test

In the 25.5 μ l final volume containing 50mM Tris (pH 7.5), 0.1mM EGTA, 0.5mM CaCl , 0.3 μ M calmodulin, 0.1% β-mercaptoethanol, 300 μ M substrate peptide, 10mM magnesium acetate and 0.02mM [ 33 P-g-ATP] (500-1000cpm/pmole), 5-20mU of CAMKKa (diluted in 50mM Tris (pH 7.5), 0.1mM EGTA, 1mg/ml BSA, 0.1% β-mercaptoethanol) for substrate peptide (AKPKGNKDYHLQTCCGSLAYRRR) is measured, and at room temperature incubated 30 minutes. Stop the assay by adding 5 μ l of 0.5M (3%) orthophosphoric acid. Use the washing buffer of 50mM orthophosphoric acid to collect the assay solution on the P81 Unifilter plate.

62.CAMKKb test

In the 25.5 μ l final volume containing 50mM Tris (pH 7.5), 0.1mM EGTA, 0.5mM CaCl , 0.3 μ M calmodulin, 0.1% β-mercaptoethanol, 300 μ M substrate peptide, 10mM magnesium acetate and 0.02mM [ 33 P-g-ATP] (500-1000cpm/pmole), measure the 5-20mU CAMKKb (diluted in 50mM Tris (pH 7.5), 0.1mM EGTA, 1mg/ml BSA, 0.1% β-mercaptoethanol) of substrate peptide (DGEFLRTSCGSPNYAARRR), and at room temperature incubate 30 minutes. Stop the assay by adding 5 μ l of 0.5M (3%) orthophosphoric acid. Use the washing buffer of 50mM orthophosphoric acid to collect the assay solution on the P81 Unifilter plate.

63.PIM1 test

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PIM1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was measured for substrate peptide (RSRHSSYPAGT) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

64.PIM3 test

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), PIM3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was measured for substrate peptide (RSRHSSYPAGT) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

65.PLK1 assay

PLK1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadate) was assayed against the substrate peptide (ISDELMDATFADQEAKKK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

66.BRSK2 trial

BRSK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was assayed against a substrate peptide (KKLNRTLSFAEPG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

67.MELK test

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 200 μM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole), MELK (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was measured for substrate peptide (KKLNRTLSFAEPG) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

68.PKCζ assay

PKCζ (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 100 μM vanadate) was assayed against the substrate peptide (ERMRPRKRQGSVRRRV) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 10 μM vanadate, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

69.Aurora C trial

Aurora C (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was assayed against the substrate peptide (LRRLSLGLRRLSLGLRRLSLGLRRLSLG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.05% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

70.ERK1 assay

5-20 mU of ERK1 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.1% β-mercaptoethanol, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

71.FGF-R1 test

FGF-R1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (poly-Glut Tyr) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

72. IRR test

The IRR of 5-20 mU to MBP (diluted in 50 mM Hepes (pH 7.5), 0.1 mM EGTA) was determined in a final volume of 25.5 μl containing 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

73.EPH-A2 test

EPH-A2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (poly-Glut Tyr) was measured in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

74.MST4 trial

5-20 mU of MST4 (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a 50 mM orthophosphoric acid wash buffer.

75.SYK test

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), SYK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) for the substrate peptide (poly-Glut Tyr) was measured and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer containing 50 mM orthophosphoric acid.

76.YES1 test

YES1 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (poly-Glut Tyr) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

77. IGF-1R test

IGF-1R (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) was assayed for substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

78. VEG-FR test

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole), VEG-FR (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) directed against the substrate peptide (KKKSPGEYVNIEFG) was assayed and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and subsequently collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

79. BTK trial

BTK (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) was measured for the substrate peptide (KVEKIGEGTYGVVYK) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

80.IR-HIS test

IR-HIS (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (KKSRGDYMTMQIG) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

81.EPH-B3 test

EPH-B3 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (poly-Glut Tyr) was measured in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

82.TBK1 (DU12569) trial

TBK1 (DU12569) (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) was assayed against the substrate peptide (KKKKERLLDDRHDSGLDSMKDEE) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 300 μM substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

83.IKKepsilon (DU14231) test

5-20 mU of IK Kepsilon (DU14231) (diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris (pH 7.5), 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-g-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid. The assay was collected onto a P81 Unifilter plate using a wash buffer containing 50 mM orthophosphoric acid.

84.GCK trial

GCK (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for MBP in a final volume of 25.5 μl and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

85. IRAK4 trial

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole), IRAK4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) against MBP was measured and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

86.NUAK1 trial

NUAK1 against ALNRTSSDSALHRRR (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM ALNRTSSDSALHRRR, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

87.MLK1 trial

MLK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for MBP in a final volume of 25.5 μl and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

88.MINK1 test

MINK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against MBP in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

89.MLK3 trial

MLK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for MBP in a final volume of 25.5 μl and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

90.LKB1 test

LKB1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.2 mM LSNLYHQGKFLQTFCGSPLYRRR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against LSNLYHQGKFLQTFCGSPLYRRR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

91.HER4 trial

In a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml poly-Glut Tyr, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole), HER4 for poly-Glut Tyr (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was measured and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

92.TTK test

TTK against RSRSRSRSRSRSRSR was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RSRSRSRSRSRSR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

93.RIPK2 trial

RIPK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for MBP in a final volume of 25.5 μl and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

94.Aurora A trial

Aurora A (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM LRRLSLGLRRLSLGLRRLSLGLRRLSLG, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against LRRLSLGLRRLSLGLRRLSLGLRRLSLG in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

95.PAK2 trial

PAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RRRLSFAEPG, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against RRRLSFAEPG in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

96.BRSK1 trial

BRSK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKLNRTLSFAEPG, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed for KKLNRTLSFAEPG and incubated at room temperature for 30 minutes in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

97.HIPK3 trial

HIPK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed for MBP in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

98.HIPK1 trial

HIPK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed for MBP in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

99.JNK3α1 assay

JNK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1% β-mercaptoethanol) directed against ATF2 (activating transcription factor) was measured in a final volume of 25.5 μl of 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, ATF2 (3 μM), 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (500-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

100.MAPKAP-K3 assay

MAPKAP-K3 against KKLNRTLSVA (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01% Brij35, 5% glycerol, 0.1% β-mercaptoethanol, 1 mg/ml BSA) was assayed in a final volume of 25.5 μl containing 50 mM β-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μM substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

101.MARK2 trial

MARK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed for KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

102.MARK4 trial

MARK4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed for KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

103.EPH-B4 test

EPH-B4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against substrate peptide (poly-GlutTyr) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

104.JAK2 trial

JAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 1 mg/ml BSA) was assayed for PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

105.EPH-A4 test

EPH-A4 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured against the substrate peptide (poly-Glut Tyr) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

106.TAK1 trial

TAK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) was assayed against the substrate peptide (RLGRDKYKTLRQIRQ) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 300 μM substrate peptide, 10 mM magnesium acetate, 0.5 mM MnCl, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

107.TrkA test

TrkA (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against the substrate peptide (poly-Glut Tyr) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MnCl, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

108. MEKK1 trial

MEKK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for MBP in a final volume of 25.5 μl and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

109.MARK1 trial

MARK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured for KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

110.CLK2 test

CLK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (RNRYRDVSPFDHSR) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.3 mM peptide, 10 mM DTT, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

111.DAPK1 assay

DAPK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) was assayed against a substrate peptide (KKLNRTLSFAEPG) in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.3 mM peptide, 10 mM DTT, 10 mM magnesium acetate, and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

112.EPH-B2 test

EPH-B2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) against the substrate peptide (poly-Glut Tyr) was measured in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid, followed by collection onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

113.TSSK1 trial

TSSK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate, and 0.02 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed against KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 mM DTT and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

114.TESK1 trial

TESK1 against actinin 2 (5-20 mU, diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.2 mg/ml actinin 2, 10 mM magnesium acetate and 0.05 mM [33P-γ-ATP] (50-1000 cpm/pmole) was measured in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 MM DTT) and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected on a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

115.TTBK1 trial

TTBK1 against RRKDLHDDEEDEAMSITA (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.3 mM RRKDLHDDEEDEAMSITA, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) was assayed in a final volume of 25.5 μl containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1% β-mercaptoethanol, 1 mg/ml BSA, 10 mM DTT and incubated at room temperature for 30 minutes. The assay was stopped by adding 5 μl of 0.5 M (3%) orthophosphoric acid and then collected onto a P81 Unifilter plate with a wash buffer of 50 mM orthophosphoric acid.

LRRK2 activity

The table below shows the IC ₅₀ values of the examples of the present invention for inhibiting LRRK2G2019S.

Table 2 _{LRRK2_IC50} values in the examples

JAK2 selectivity

The table below shows the JAK2 _IC50 values of Examples of the present invention.

_{JAK2_IC50} values in Table 1

Activity in Drosophila models

Survival rate

The following table shows the survival rates of the examples of the present invention.

Example Survival rate 29 * 58 **

**P < 0.01, compared with DMSO negative control;

*P<0.05, compared with the DMSO negative control.

Climbing test

The following table shows the climbing measurement of an embodiment of the present invention.

**P < 0.01, compared with DMSO negative control;

*P < 0.05, compared with DMSO negative control;

Kinase assay at week 6

The following table shows the results of kinase assays according to the examples of the present invention.

*P<0.05, compared with the DMSO negative control.

Kinase selectivity data

Kinase selectivity data for representative compounds are shown in the table below. Values are expressed as percent inhibition of each specific kinase at 1 μM inhibitor concentration.

Table 1 Kinase selectivity data of representative compounds

Various modifications and variations of the described aspects of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present invention, which modifications will be apparent to those skilled in the relevant art, are intended to be within the scope of the appended claims.

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Claims (11)

1. A compound of general formula I or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof: in: V is N; W is N; ^R1 is selected from the following groups: Y is selected from the following groups: ^X1 is -( _CH2 ) _n- ; Z is -( _CH2 ) _n- ; n is 1, 2, or 3. 2. A compound, wherein the compound has one of the following structures or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof: 22, 23, 29, 55, 68. 3. A pharmaceutical composition comprising the compound according to claim 1 or 2. 4. The pharmaceutical composition according to claim 3, further comprising a pharmaceutically acceptable excipient or a combination thereof. 5. The pharmaceutical composition according to claim 3 or 4, further comprising a second therapeutic agent. 6. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of a medicament for treating a condition caused by at least one of cancers and neurodegenerative diseases associated with LRRK kinase. 7. The use according to claim 6, wherein the neurodegenerative disease is Parkinson's disease. 8. Use of the compound according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 3 to 5 in the preparation of a medicament for the prevention or treatment of a condition caused by, associated with or accompanied by, abnormal LRRK kinase activity. 9. The use according to claim 8, wherein the LRRK kinase is an LRRK2 kinase. 10. Use in a test of the compound according to claim 1 or 2 or the pharmaceutical composition according to any one of claims 3 to 5, wherein the test is used to identify additional candidate compounds capable of inhibiting LRRK kinase. 11. The use according to claim 10, wherein the LRRK kinase is an LRRK2 kinase.