Development of amino-pyrimidine inhibitors of c-Jun N-terminal kinase (JNK): Kinase profiling guided optimization of a 1,2,3-benzotriazole lead

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1486–1492

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Development of amino-pyrimidine inhibitors of c-Jun N-terminal kinase (JNK): Kinase profiling guided optimization of a 1,2,3-benzotriazole lead Wylie S. Palmer a,⇑, Muzaffar Alam a, Humberto B. Arzeno a, Kung-Ching Chang a, James P. Dunn a, David M. Goldstein a, Leyi Gong a, Bindu Goyal a, Johannes C. Hermann a, J. Heather Hogg a, Gary Hsieh a, Alam Jahangir a, Cheryl Janson b, Sue Jin a, R. Ursula Kammlott b, Andreas Kuglstatter a, Christine Lukacs b, Christophe Michoud b, Linghao Niu a, Deborah C. Reuter a, Ada Shao a, Tania Silva a, Teresa A. Trejo-Martin a, Karin Stein a, Yun-Chou Tan a, Parcharee Tivitmahaisoon a, Patricia Tran a, Paul Wagner a, Paul Weller a, Shao-Yong Wu a a b

Roche Palo Alto, 3431 Hillview Ave., Palo Alto, CA 94304, USA Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA

a r t i c l e

i n f o

Article history: Received 19 November 2012 Revised 12 December 2012 Accepted 13 December 2012 Available online 22 December 2012 Keywords: c-Jun N-terminal kinase (JNK) CDK Kinase inhibitor Kinase selectivity

a b s t r a c t A series of amino-pyrimidines was developed based upon an initial kinase cross-screening hit from a CDK2 program. Kinase profiling and structure-based drug design guided the optimization from the initial 1,2,3-benzotriazole hit to a potent and selective JNK inhibitor, compound 24f (JNK1 and 2 IC50 = 16 and 66 nM, respectively), with bioavailability in rats and suitable for further in vivo pharmacological evaluation. Ó 2013 Elsevier Ltd. All rights reserved.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family1 and are primarily activated by cytokines and environmental stress.2,3 The JNKs are serine/threonine protein kinases that are able to phosphorylate the N-terminal transactivation domain of c-Jun, resulting in enhancement of c-Jun dependent transcriptional events. There are three closely related JNK genes that are expressed as 10 different isoforms by mRNA alternative splicing.4,5 While JNK1 and JNK2 are ubiquitously expressed, JNK3 is primarily present in the brain, and reduced levels in cardiac muscle, and testis.6 Based upon the role of JNK in regulating members of the activator protein-1 (AP-1) transcription factor as well as other cellular factors implicated in gene expression, cellular survival and proliferation, inhibiting JNK may have many potential therapeutic utilities.7,8

Abbreviations: JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; CDK-2, cyclin-dependent kinase-2. ⇑ Corresponding author at present address: Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110, USA. E-mail address: [email protected] (W.S. Palmer). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.12.047

Consequently, many types of JNK inhibitors have been reported in the literature.9–15 JNK plays an important role in the T cell immune response and regulates the expression or function of a number of pro-inflammatory cytokines (TNFa, IL-2, IL-6, etc.) that are central to many human inflammatory disorders.16 Our interest is in the role of JNK for rheumatoid arthritis (RA) and evidence suggests that both JNK1 and JNK2 are important for immuno-modulatory function,17 therefore a dual JNK inhibitor was sought. We wish to report here our initial discovery and development of a series of amino-pyrimidines into selective and potent JNK inhibitors with suitable pharmacokinetic properties for further in vivo evaluation. The initial hit, compound 1 (Fig. 1), originated from a cyclin-dependent kinase-2 (CDK2) program and identified as a JNK inhibitor by kinase cross-screening. Compound 1 was an attractive lead because it already showed some selectivity for JNKs versus the CDKs, and no inhibition against p38. The transcyclohexyl group also differentiated it from closely related CDK inhibitors.18 Encouraged by this initial selectivity, potency, low molecular weight (MW = 387), and low lipophilicity (c Log P = 2.3) we initiated lead optimization. We used structure-based drug

W. S. Palmer et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1486–1492

N HN

N

N

N

N

NHSO 2Me Figure 1. Initial hit from kinase cross-screening, compound 1 (IC50: JNK1 24 nM, JNK2 97 nM, JNK3 114 nM, CDK1 1.52 lM, CDK2 0.30 lM, CDK4 0.91 lM, p38 >10 lM).19

Table 1 Biological effect of benzotriazoles20 with cyclohexyl-amine substituents

N N

X Compound

1 2 3 4 5 6 7 8

N N

N

N

N

N

1-6

X

O

N

N

N

7, 8

IC50a (lM)

X

NHSO2Me H OH NH2 NHSO2NMe2 NH(C@O)CH3 1-Pyrrolidinyl 1-Morpholino

N

JNK1

JNK2

CDK2

c-Junb

HCT116c

0.024 0.10 0.029 0.074 0.019 0.039 0.12 0.063

0.097 0.39 0.11 0.17 0.076 0.23 0.40 0.18

0.30 2.7 0.43 0.36 1.7 0.57 >6.2 2.0

1.3 nt 0.79 0.54 4.9 1.5 3.3 1.8

2.3 nt 1.2 0.77 22 16 >30 20

a

Values are a mean of at least three experiments. JNK cell assay measuring inhibition of phospho-c-Jun in SW1353 cells.15 c Cell viability assay measuring reduction of WST-1 reagent in an HCT-116 cellline (nt = not tested). b

design, in vitro safety assays and kinase profiling in order to guide the optimization of this initial lead. We evaluated the biological effect of various cyclohexyl-amine substituted 1,2,3-benzotriazoles20 (compounds 1–8) on cell and enzyme potencies, as well as their effect on cytotoxicity (Table 1).

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The compounds were screened in JNK1, JNK2 and CDK2 enzyme assays19 and JNK cell potencies were evaluated by measuring the TNFa-induced phosphorylation of the downstream c-Jun substrate.15 Cytotoxicity was evaluated by measuring the reduction of WST-1 reagent in an HCT-116 cell proliferation assay. This assay is also sensitive towards CDK inhibition and gives a cellular readout for CDK inhibition. In general, cyclohexyl-amine substituents improved upon the potency of JNK1 and JNK2 relative to the unsubstituted cyclohexane 2, and compounds were consistently four to fivefold more potent for JNK1 versus JNK2. Sub-micromolar JNK cell potency could be achieved, as in the case of the alcohol 3 and amine 4, however, neither had a window with respect to cytotoxicity. Other compounds, such as 5 and 6, demonstrated better cytotoxicity windows (10-fold in the case of 6) but were unselective with respect to CDK2 activity. The most promising sub-series, represented by amides 7 and 8, showed better JNK versus CDK enzyme selectivity and maintained reasonable cell potencies with greater than 10-fold cytotoxicity windows. The cyclohexyl-amine substituents also affected the compounds’ selectivity towards the kinome. Compounds were profiled at 10 lM against a diverse panel of 128 kinases using an in vitro ATP-site competition binding assay.21 The kinase profiles22 shown in Figure 2 demonstrated that the basic amine 4 (A) interacted with more kinases than the morpholino-amide 8 (B), with calculated selectivity scores23 of S15 = 0.31 and 0.17, respectively. The selectivity score represents the fraction of kinase interactions, whereby S15 is the number of non-mutant kinases with less than 15% of control activity (or greater than 85% binding activity) divided by the total number of non-mutant kinases tested. The higher the score, the more promiscuous the compound, and in general, we observed that higher promiscuity correlated with increased cytotoxicity, as exemplified by 4. The improved selectivity for JNK relative to CDK2 displayed by amides 7 and 8, as well as the improved kinome selectivity demonstrated by 8, provided the rationale for future exploration of amide-substituents in this position of the molecule (vide infra). When we investigated the structure–activity relationships (SAR) for the benzotriazoles, it became evident that a significant proportion of analogues inhibited cytochrome P450 (CYP) enzymes, particularly the 2C9 isoform. For example, compounds 3 and 6 were both potent CYP 2C9 inhibitors (IC50 values of 7.8 and 39 nM, respectively). We suspected that the 1,2,3-benzotriazole

Figure 2. Kinase profiles for amine 4 (A) and morpholino-amide 8 (B), against a panel of 128 kinases.21 Compounds were tested at 10 lM compound concentration. Profiles were generated with TREEspot software tool with 15% cutoff.22 The size of the spot represent % interaction with a particular kinase, blue dots are JNK1, 2, and 3; red dots are off-target kinases. Selectivity scores,23 S15: 4 = 0.31, 8 = 0.17.

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Table 2 Biological activity of heterocyclic replacements

N N

OH

Compound

N

N

N

*

*

*

N

0.029

0.11

0.79

0.57

1.2

12

0.087

0.25

nt

0.055

0.35

2.7

0.065

0.36

2.0

0.25

0.65

2.3

0.14

0.72

8.2

0.77

2.9

nt

N

N

12

N

*

NH

13

*

NH

14

*

c-Junb

N

11

*

JNK2

N

N

10

JNK1

N

N

9

a

9-15 IC50a (lM)

3

b

R

R-group

*

15

N

N

Values are a mean of at least three experiments. nt = Not tested. JNK cell assay measuring inhibition of phospho-c-Jun in SW1353 cells.15

group was responsible for potent CYP inhibition since 1amino-benzotriazole is a known CYP inactivator.24 Therefore, part of our optimization efforts included finding a heterocyclic replacement for the benzotriazole. Table 2 summarizes the biological activity of a few of the heterocycles that were evaluated as replacements for the 1,2,3-benzotriazole. The 4-trans-aminocyclohexanol derivatives were synthesized25 and used as a means for comparing the different heterocycles. It was quite evident that the number of hetero-atoms and their placement within the template have a pronounced and unpredicted effect on enzyme and cellular potencies. For example, the 1,2,3-benzotriazole 3 is at least 10-fold more potent than the 1,2,4-triazolopyridine 9, while imidazopyridine 1026 is nearly as potent as 3. Also, the N-linked benzimidazole27 was significantly less active than 10, however, N-linked heterocycles (11 and 12) were two- to fourfold more potent than their corresponding Clinked analogues (13 and 14, respectively). It was also evident that co-planarity of the heterocycle with the pyrimidine was important

as demonstrated by the loss in potency of both indoline 15 and a 2methylindole27 compared to indole 12. Small hetero-atom changes within the heterocycle also unexpectedly had a profound effect on kinome selectivity. Kinase profiles for four compounds against a panel of at least 227 kinases at 10 lM compound concentration are shown in Figure 3. The profiles show a reverse correlation between decreasing number of nitrogens within the heterocycle and increasing kinome selectivity. For example, compare benzotriazole 3 (A), indazole 11 (B) and indole 12 (C) which follow a descending selectivity order with S15 values of 0.26, 0.19, and 0.10, respectively. Interestingly, the 3linked indazole 13 (D) was significantly less selective (S15 0.51) than its regioisomer 11 and interacted with over half of the kinases within the panel. A likely explanation for this is that there is an alternative hinge-binding mode with the two-indazole nitrogens of 13 that is not possible with 11.28 Encouraged by the increased kinome selectivity, lack of CYP liabilities27 and reasonable cell potencies of 11 and 12, we chose to further develop these heterocycles. Further improvements in JNK potency were guided by X-ray cocrystal structures. Compound 11 bound to JNK1b enzyme (Fig. 4) shows the pyrimidine nitrogen and hydrogen of the cyclohexylamine forming the classical bi-dentate interaction with hinge residues M111 and L110, respectively. The indazole is co-planar with the pyrimidine and the cyclohexyl-group positions the alcohol towards solvent where further changes could be made in order to improve the physicochemical properties of the molecule as well as impart kinase selectivity as we had observed for the benzotriazole series. We surmised that potency could be gained by filling space underneath and making additional interactions with the glycinerich loop. We targeted further functionalization from the 4-position of the indazole/indole and initial SAR work led to a series of 4-alkoxy substituents which were synthesized by the methods outlined in Schemes 1 and 2, respectively.25 The indazoles 18 and 19 (Scheme 1) were synthesized by nucleophilic displacement of 4-chloro-2-thiomethylpyrimidine by hydrazine, followed by cyclo-condensation with 2-fluoro-6-methoxy-benzaldehyde to give intermediate 16. Boron tribromide deprotection of methyl-ether 16, followed by alkylation with the appropriate electrophile provided intermediate 17. Final products, 18 and 19, were synthesized by two different methods: a two-step procedure using MCPBA to oxidize the methyl-sulfide of 17 to form either the sulfoxide or sulfone, followed by nucleophilic displacement of the amine, or by a one-pot procedure using NCS as the oxidant. Although, the initial medicinal chemistry route towards indole derivatives 21–2425 also started with 4-chloro-2-thiomethylpyrimidine, our process research group developed an improved synthesis as outlined in Scheme 2.29 The propoxy-sulfone group is first installed onto 4-hydroxyindole using standard Finkelstein displacement conditions. A regioselective displacement of 2,4dichloropyrimidine was then performed with the indole intermediate using HOBT (20 mol %), providing the 4-substituted indole 20 in excellent yield (95%). The HOBT additive was essential for achieving high regioselectivity and yields for this transformation, since standard displacement conditions for this transformation using sodium hydride in DMF gave a 30% yield as a 3:1 mixture of 4- versus 2-substituted products. We could not isolate or identify an intermediate in this transformation, but we postulate that a more reactive 4-substituted HOBT adduct is initially formed and responsible for the improved selectivity. Nucleophilic displacement of 20 by the appropriate amine gave 21 and 22. Hydrolysis of 22 and coupling of carboxylic acid 23 with various amines provided amides 24a–f. The biological activity of 4-alkoxy substituted compounds is summarized in Table 3. The effect of substituent modifications

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Figure 3. Kinase profiles for 1,2,3-benzotriazole 3 (A), 1-indazole 11 (B), and 1-indole 12 (C), against a panel of 317 kinases and 227 kinases for 3-indazole 13 (D). Compounds were tested at 10 lM compound concentration. Profiles were generated with TREEspot software tool with 15% cutoff.24 The size of the spot represents % interaction with a particular kinase, blue dots are JNK1, 2, and 3; red dots are off-target kinases. Selectivity scores, S15: 3 0.26, 11 0.19, 12 0.10, 13 0.51.

Figure 4. X-ray crystal structure of compound 11 with JNK1b enzyme (2.4 Å resolution, PDB accession number 4HYS).

on compound protein binding was evaluated by a JNK2 enzyme protein-shifted assay, whereby protein shift was calculated by dividing the JNK2 IC50 measured in the presence of 40% human

serum by the JNK2 IC50 value without human serum. Although potency was increased by the addition of a propoxy substituent (18b), cellular potency was lost, presumably due to high protein binding. The addition of polar substituents, such as alcohols and sulfonamides improved the physical properties of the molecule and cellular activity was regained. A sulfone-group, and particularly, the propoxy-sulfone of 18g proved to be optimal for increasing JNK enzyme potencies into the nanomolar range. While both indazole and indole-series were concurrently optimized, the lower protein shifts exhibited by the indoles (compare 19 and 21) gave impetus to further optimize this series. The increasing potency gained by the addition of substituents in the 4-position is nicely rationalized by the X-ray crystal structure of compound 18g complexed with JNK1b (Fig. 5). The propoxysulfone moiety nicely fills the space underneath the glycine-rich loop and each oxygen of the sulfone is H-bonded to residues K55 and Q37. Because of the improved kinome selectivity previously demonstrated by amides 7 and 8, a series of amides were also evaluated on the indole series. Table 4 summarizes the biological activity, protein-shifts and in vitro human liver microsomal clearance of compounds 24a–f. Although, the addition of amides provided the low JNK2 protein-shifts that we desired, amides, such as diethylamide 24a, were predicted to have rapid clearance. Interestingly, microsomal stability was only modestly improved with the

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N S

N

i N

S

Cl

N

ii N

NHNH2

S

O

N

O

F

N

N

16 O

N S

iii, iv

16

N N

N

N

v, vi O

R

HN

N

N

N

or vii O

17

R

X 18 a-g: X = OH 19: X = C(CH3)2OH

Scheme 1. Reagents and conditions: (i) hydrazine, K2CO3, ethanol, reflux, 58%; (ii) 5 equiv NaOH in EtOH, 80 °C, 95%; (iii) BBr3, 92%; (iv) R–X (X = Br or tosylate), NaH or K2CO3, 40–91%; (v) MCPBA in CH2Cl2, 0 °C, 2 h, 95–100%; (vi) excess amine, 80–140 °C in NMP, 13–59%; (vii) NCS, water/NMP, 80–90 °C for 20 min, then excess amine, 10– 52%.

HN

N

i, ii

N

N

Cl

N

iii

OH

O

HN

iv

O X

23 X = CO2H

N

SO2Me

20

22 X = CO2Et

N

v

SO2 Me

21 X = C(CH3)2OH 22-24

24a-f X = CONR2

Scheme 2. Reagents and conditions: (i) 1-chloro-3-(methanesulfonyl)-propane, K2CO3, KI in acetonitrile, 80 °C, 82%; (ii) 2,4-dichloropyrimidine, HOBT (20 mol %), K2CO3, in DMA, 85 °C, 95%; (iii) amine HCl salt, K2CO3, NMP, 80 °C; 97% for 22; (iv) NaOH, IPA/water 80 °C, 97%; (v) amine, BOP or HBTU, Et3N, 69–99%.

Table 3 Biological activity of 4-alkoxy-substituted indazoles (18a–g, 19) and indole (21)

N

N HN

N

N

N

HN O

OH

N

N

R

18 a-g

X

O OH

SO2Me

19: X = N 21: X = CH

Compound

R-group

JNK1/2 IC50a (nM)

JNK2 Fold-shiftb

c-Junc IC50a (lM)

18a 18b 18c 18d 18e 18f 18g 19 21

CH3 CH2CH2CH3 CH2CH2CH2OH CH2CH(OH)CH2OH CH2CH2CH2NHSO2CH3 CH2CH2SO2CH3 CH2CH2CH2SO2CH3 na na

23/178 12/79 24/139 34/194 56/246 33/151 6.2/24 4/21 4/27

37 147 28 5.7 17 4 23 18 6.3

3.1 >30 0.96 2.0 1.1 0.98 1.0 0.24 0.35

Figure 5. X-ray crystal structure of compound 18g with JNK1b enzyme (2.15 Å resolution, PDB accession number 4HYU).

a

Values are a mean of at least three experiments. Fold-shift = JNK2 enzyme IC50 with 40% human serum divided by JNK2 enzyme IC50 without human serum. c JNK cell assay measuring inhibition of phospho-c-Jun in SW1353 cells.15 b

mono- and difluoro-substituted derivatives, 24b and 24c. Alcoholsubstituents proved optimal, as 24e and 24f were metabolically stable, had low protein-shifts and good cellular potency. Compound 24f was profiled against a panel of 317 kinases at 10 lM and 43 Kd values were determined for the kinases which

showed greater than 85% binding interaction (S15 = 0.15). Kd values for the 16 most potent kinases as well as two P38 isoforms are shown in Table 5 in descending order of binding affinity. Kinase selectivity for 24f is good with respect to JNK1 (160-fold difference between JNK1 and the next most potent kinase PRKR). The activity of 24f is not dependent upon the P38 pathway since selectivity against P38a and P38b is excellent and 24f did not inhibit the P38-dependent phosphorylation of HSP27 (IC50 >30 lM) in a cellular assay.

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W. S. Palmer et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1486–1492 Table 4 Biological activity of indole amides 24a–f

Table 6 In vitro and in vivo properties of compound 24f

N HN

In vitro

N

N SO2 Me

O X Compd

O

X-group

JNK2 Fold-shiftb

c-Junc IC50 (lM)

14/57

4.6

0.58

609

14/64

2.5

0.59

175

16/51

4.2

1.4

283

4.7/28

2.9

1.4

141

9.7/47

2.6

1.1

19

JNK1/2 IC50 (nM)

N

24a

24 a-f

a

HLMd a

N 24b

F 24c

N

F

Aq solubilitya (lg/mL)

Cacob AB/BA

Protein bindingc

3.9

0.2/11.5

5% Free

Microsomal stabilityd Clint (human) Clint (rat)

In vivo rat (male, Hanover–Wistar)

e

Route

Dose (mg/ kg)

Cl (mL/ min/kg)

T1/2 (h)

Cmax (ng/ mL h)

AUC0–inf (ng/ mL h)

ivf pog pog

0.5 2 20

4.6

2.6 4 2.9

2670 91 3680

1850 490 9330

30 52

%F

7 13

a

Equilibrium aqueous solubility in phosphate buffer at pH 6.5, 25 °C. Permeability in Caco-2 cells, AB (apical to basolateral) and BA (basolateral to apical) movement of 10 lM test compound in 21 days cultured Caco-2 cells (cm/ s  10E 6), pH 7.4. c CentrifreeÒ, human plasma. d Liver microsomal intrinsic clearance (lL/min/mg protein). e Tested in triplicate. f Solution formulation: 5% DMSO, 80% PEG400, 10% water, 5% NaCl (v/v). g Aqueous solution formulation, pH 3.5. b

F

N 24d

O N 24e

HO

Supplementary data

N 24f

16/66

1.8

1.1

30

HO a

Values are a mean of at least three experiments. Fold-shift = JNK2 enzyme IC50 with 40% human serum divided by JNK2 enzyme IC50. c JNK cell assay measuring inhibition of phospho-c-Jun in SW1353 cells.15 d In vitro human liver microsomal clearance (lL/min/mg). b

Table 5 Kinase selectivity profile of 24fa

a

suitable for further in vivo pharmacological evaluation. To date, there have been no successful JNK compounds in the clinic, but hopefully advanced compounds such as 24f will prove useful in unraveling the many roles JNK enzymes play in disease.

Kinase

Kda

Kinase

Kda

Kinase

Kda

JNK1 JNK3 JNK2 PRKR MEK6 ERK3

0.00075 0.0018 0.013 0.12 0.31 0.33

DAPK1 MEK4 DRAK1 STK16 ADCK CDK7

0.38 0.39 0.44 0.45 0.49 0.62

SgK085 ERK8 LIMK1 DAPK3 P38b P38a

0.62 0.65 0.87 0.88 3.2 8.8

Kd for kinase binding in lM. See Supplementary data for all 43 Kd values.

Compound 24f represented an advanced compound for the program and showed reasonable pharmacokinetic properties for further in vivo pharmacological evaluation (Table 6). Although, 24f had a high transporter efflux ratio of 50 (Caco BA/AB), its intrinsic permeability was reasonable (Caco AB with Elacridar 2.4  10E 6 cm/s) and demonstrated low clearance in vivo. Presumably efflux transporters could be saturated in rats since we observed an increased bioavailability of 13% at the higher 20 mg/kg dose. This compound allowed us to measure the effect of JNK inhibition in an in vivo rheumatoid arthritis model and results will be reported in due course. In conclusion, kinase profiling not only provided the initial starting point for developing a series of indole-substituted amino-pyrimidines, but was also important for guiding the optimization of the series to give a potent and selective JNK inhibitor

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18. Byth, K. F.; Culshaw, J. D.; Green, S.; Oakes, S. E.; Thomas, A. P. Bioorg. Med. Chem. Lett. 2004, 14, 2245. 19. In vitro JNK1 and JNK2 enzyme activity was measured by phosphorylation of GST-ATF2 (19–96) with [c-33P] ATP. In vitro CDK2 enzyme activity was measured by phosphorylation of a 6-histidine tagged truncated form of retinoblastoma (Rb) protein (amino acids 386–928) with [c-33P] ATP. Detailed assay conditions can be found in Ref. 20. 20. Representative experimental procedures for compounds 1–8 are described in US20080103142. 21. Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329. 22. Image generated using TREEspot™ Software Tool and reprinted with permission from KINOMEscan™, a division of DiscoveRx Corporation, Ó DISCOVERX CORPORATION 2010. Note: atypical, lipid, pathogen and mutant kinases are not depicted on dendrogram, see Supplementary data for full data set.

23. Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. Nat. Biotechnol. 2008, 26, 127. The selectivity score calculation is also described at: http:// www.discoverx.com/tools-resources/leadhunter-study-reports-data-analysis. 24. Ortiz de Montellano, P. R.; Mathews, J. M. Biochem. J. 1981, 195, 761. 25. Representative experimental procedures can be found in US20080146565. 26. WO2010097335 F. Hoffmann-La Roche, Switzerland. 27. Data not shown. 28. Alternative binding modes have been reported for an imidazopyridine analogue similar to compound 10, see: Buckley, G. M.; Ceska, T. A.; Fraser, J. L.; Gowers, L.; Groom, C. R.; Higueruelo, A. P.; Jenkins, K.; Mack, S. R.; Morgan, T.; Parry, D. M.; Pitt, W. R.; Rausch, O.; Richard, M. D.; Sabin, V. Bioorg. Med. Chem. Lett. 2008, 18, 3291. 29. U.S. Patent 8183,254, May 22, 2012.