Targeted polypharmacology: Discovery of dual inhibitors of ......1 Targeted polypharmacology: Discovery of dual inhibitors of tyrosine and phosphoinositide kinases Beth Apsel1, Jimmy

1

Targeted polypharmacology: Discovery of dual inhibitors of tyrosine and phosphoinositide kinases Beth Apsel1, Jimmy A. Blair2, Beatriz Z. Gonzalez3,4, Tamim M. Nazif5, Morri E. Feldman1, Brian Aizenstein6, Randy Hoffman6, Roger L. Williams3, Kevan M. Shokat2,5, and Zachary A. Knight5,7

1 Program in Chemistry and Chemical Biology, University of California, San Francisco

2 Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720 3 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 4 Present Address: Instituto de Química-Física “Rocasolano” (CSIC), Serrano 119, 28006 Madrid, Spain.

5 Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco

6 Invitrogen Corporation, 501 Charmany Drive, Madison, WI 53719

7 Present Address: The Rockefeller University, 1230 York Ave., New York, NY 10021 Correspondence: [email protected]

PP

102

PP

131

PP

129

PP

133

PP

118

PP

130

PP

132

PP

139

PP

146

PP

142

PP

140

PP

147

PP

105

PP

158

0.0010.010.1110

100

N

N N NN

Me NMe

NCl

NNH2

NNH2

NN

N NN NHN

ON

NHNH2

HNO

NHNNH2

NMe

N NNHN

NHHN

NHNNHN

NHNH2NNO

NH2

PP

160

PP

136

PP

138

PP

94

PP

121

PP

124

PP

154

PP

115

PP

159

PP

157

PP

162

NHN

FNH

OH

PP

43

Supplementary Figure 1

a

b

mTORDNA-PK

SrcVEGFR

p110α

R1NN N

N

NH2 R1

IC50 (μM)

R1

R2

NN N

N

NH2 R1

R2

0.0010.010.1110

IC50 (μM)

100

Me

NH

NHH

NH2

NH2O

O

0.010.1

110

1001000

NH

p110α

0.11

10100

100010000 mTOR

0.1

1

10

100DNA-PK

Src

0.010.1

10100

1

VEGFR2

Me

NH

NHH

NH2

NH2O

O

NH

Me

NH

NHH

NH2

NH2O

O

NH

Me

NH

NHH

NH2

NH2O

O

NH

Me

NH

NHH

NH2

NH2O

O

NH

IC50IC50

R2R2

R2

0.010.1

110

1001000

IC50IC50

R2R2

R2

IC50IC50

R2R2

IC50IC50

R2R2

IC50IC50

R2R2

R2 R2

R2

Supplementary Figure 1. Structure-activity relationship data for selected inhibitors. a, IC50 values are plotted on the y-axis for inhibitors from each chemical series against two tyrosine kinases (Src and VEGFR2) and three PI3-K family members (p110α, mTOR and DNA-PK). Chemical structures are represented on the x-axis. b, The y-axis plots the ratio of the IC50 values for a given R1 substituent when R2 equals either the substituent listed on the x-axis or isopropyl. This provides a graphical depiction of the propensity of different R2 substituents for binding to different kinases in the context of diverse R1 substituents. For example, isopropyl and cyclopentyl were the optimal R2 groups for all three PI3-K family members (average ratio ≈ 1; p110α, mTOR and DNA-PK), whereas cyclopentyl was favored over isopropyl for the tyrosine kinases Src and VEGFR2 (average ratio < 1). Among tyrosine kinases and mTOR, there was a steep penalty for decreasing the size of the R2 substituent to smaller than isopropyl. By contrast, there was little penalty for decreasing the size of the R2 substituent for binding to DNA-PK. Basic groups were strongly disfavored by all kinases. For molecules chiral at R2, the differences in IC50 values between enantiomers was small, and therefore the data for both stereoisomers is pooled in this figure.

pAkt (308)pAkt (473)

pS6K (389)pERK1/2

pS6S6

+ + + + + +

-

-

-

Serum (10%)

[PP242] (μM) 10 2.5

0.62

50.

156

0.03

9


Supplementary Figure 2. PP242 directly inhibits mTOR and blocks Akt phosphorylation. BT549 breast cancer cells were serum starved overnight then stimulated with 10% serum in the presence of PP242 (0.040 to 10 μM) for 30 min. Cells were lysed and phosphorylation of signalling proteins was probed by western blotting. pS6 (Ser235/236), pERK (Thr202/Tyr204).

90o

PP121

PP121

Glu310

Asp841


Src

p110γ

Asp841p110γ

Glu310Src

Supplementary Figure 3. Structural model of PP121 bound to c-Src and p110γ. Comparison of the hydrogen bond between PP121 (orange) and Glu310 (orange) observed in the c-Src/PP121 co-crystal structure and a model of an analogous hydrogen bond between PP121 (gray) and Asp841 (gray) in p110γ. Modelling based on the co-crystal structure between p110γ and S1.

[Drug] (μM)

tubelength

(%)

DMSO

10 μM

2.5 μM

0.625 μM

0.156 μM

0.040 μM

00

255075

100

0.1 1 10

PP121

PP121 sorafenib PIK-90PI-103PP102

d

PP102PP121

0255075

100

0 0.1 1 10

PP121

PI-103PP121

0 0.1 1 100

255075

100

[Drug] (μM) [Drug] (μM) [Drug] (μM) [Drug] (μM)

sorafenibPP121

0 0.1 1 100

255075

100

PIK-90PP121

0 0.1 1 100

255075

100

sorafenibPIK-90PI-103PP102

c


a

b

-10 -9 -8 -7 -6 -5 -4

0255075

100125

log [PP102] (M)

SerumVEGF

Cel

l pro

lifer

atio

n (%

)

-10 -9 -8 -7 -6 -5 -4

0255075

100125

log [PP121] (M)

SerumVEGF

Cel

l pro

lifer

atio

n (%

)

pAkt (308)pVEGFR2

pAkt (473)pERK1/2

pS6S6

+ + + + + +VEGF

-(μM)

[PP121] 10 2.5

0.62

50.

156

0.04

-

- + + + + + +VEGF

-(μM)

[PP102] 10 2.5

0.62

50.

156

0.04

-

-

pAkt (308)pVEGFR2

pAkt (473)pERK1/2

pS6S6

Supplementary Figure 4. PP121 directly inhibits VEGFR2 and in vitro endothelial cell angiogenesis. a, HUVECs were serum starved overnight and then stimulated with VEGF (4 ng/mL) in the presence of PP102 or PP121 (0.040 to 10 μM) for 30 min. Cells were lysed and probed by western blotting. pVEGFR2 (pTyr1175). b. Proliferation of endothelial cells in the presence of PP102 or PP121 (0.040 to 10 μM) in either complete media (serum) or basal media supplemented with VEGF (4 ng/mL). c, Quantitation of endothelial cell tube formation in the presence of selected inhibitors. d, Brightfield and Calcein AM stained (inset) endothelial cells following tube formation assay in the presence of selected inhibitors.

0 1 2 3 4 5 6 7 8 90

1000200030004000

50009000

13000

Time (d)

Cel

l Num

ber

DMSOPIK-90PP102

PI-103

ImatinibPP121

150 250

100 75 50 37

150 250

100 75 50 37

pTyr

pAkt (308)

pS6pAkt (473)

actin

150 250

100 75 50 37

a

b


c

IC50

3 6 9

0.01

0.1

1

10

Day

(μM

)P

rolif

erat

ion

PIK-90PP102

PI-103

ImatinibPP121

-(μM)

[PP121] 20 5.0

1.25

0.31

0.08

-(μM)

[PI-103] 20 5.0

1.25

0.31

0.08

-(μM)

[Imatinib] 20 5.0

1.25

0.31

0.08

pTyr

pAkt (308)

pS6pAkt (473)

actin

pTyr

pAkt (308)

pS6pAkt (473)

actin

Supplementary Figure 5. PP121 directly inhibits Bcr-Abl in K562 cells. a, K562 cells were treated with the indicated concentration of each inhibitor (2 h), lysed, and blotted for indicated proteins. Molecular weights indicated adjactent to phosphotyrosine (pTyr) blots. b, Proliferation of K562 cells in response to selected drugs (2.5 μM). c, IC50 values for inhibition of K562 cell proliferation by selected drugs.

Supplementary Table 1Name PP200 PP201 PP1 PP204 PP205 PP206NCB # 200 201 9 204 205 206

R1

R2 H H H H H

p110α 8.6 8.5 20 4.2 15 9p110β 100 50 100 23 100 50p110δ 12.5 9.6 7.5 5.7 29 11p110γ 10.9 7.7 100 2.4 25 15mTOR ND ND 1.1 ND ND NDDNAPK 1.6 1.3 1 1.1 7.1 3.7PI4Kβ ND ND ND ND ND ND

Abl ND ND 0.158 ND ND NDHck ND ND 0.012 ND ND NDSrc ND ND 0.0064 ND ND ND

Src (T/I) ND ND 1.5 ND ND NDVEGFR ND ND 0.103 ND ND NDEGFR ND ND 0.15 ND ND NDEphB4 ND ND 0.171 ND ND ND

Name PP125 PP56 PP207 PP208 PP209 S1NCB # 125 56 207 208 209 7

R1

pyrrolopyrimidine N-Me exocyclic

R2 H H

p110α 19 0.51 4.1 3.8 ND 0.48p110β 100 0.38 25 50 100 8.6p110δ 18 0.084 3.1 2.9 100 0.25p110γ 15 0.68 1.7 6.4 100 1.5mTOR ND 6 ND ND ND 2.1DNAPK 5 0.62 0.44 0.25 ND 0.19PI4Kβ ND 1.6 ND ND ND 2.6

Abl ND 1.7 ND ND ND 0.07Hck ND 0.047 ND ND ND 0.023Src ND 0.075 ND ND ND 0.015

Src (T/I) ND 3.2 ND ND ND 3.1VEGFR ND 1.45 ND ND ND 0.213EGFR ND 0.922 ND ND ND 0.954EphB4 ND 1.23 ND ND ND 0.316

OHOH OH OH

MeMe Me

OH

Me Me

Me

Supplementary Table 1 continuedName PP60 PP318 PP320 PP333 PP323 PP327NCB # 60 318 320 333 323 327

R1

R2 H H Me

p110α 1.35 14.7 1.5 0.64 0.28 0.75p110β 4.23 100 5.87 3.13 1.72 5.29p110δ 0.294 3.19 0.66 0.21 0.22 0.47p110γ 1.31 3.34 0.852 0.55 0.435 0.772mTOR 0.15 1.2 4 0.861 0.033 0.016DNAPK 0.316 24.3 0.478 0.173 0.212 0.336PI4Kβ 1.36 31 8.8 100 12 40

Abl 2.18 13 0.56 0.233 0.031 0.16Hck 0.033 12.7 0.078 0.065 0.0051 0.019Src 0.066 33 0.164 0.096 0.0043 0.016

Src (T/I) 0.821 20.3 10.6 9.97 0.67 1.56VEGFR 9.63 100 0.237 0.151 0.01 0.043EGFR 100 ND 12.4 19 0.663 1.67EphB4 1.16 12.2 18.3 8.93 0.249 1.02

Name PP77 PP78 PP22 PP79 PP85 PP87NCB # 77 78 508 79 85 87

R1

R2

p110α 2.01 1.27 0.175 0.266 6.31 0.802p110β 5.81 5.61 0.35 0.343 50.3 0.255p110δ 0.677 0.249 0.046 0.072 2.97 0.13p110γ 1.47 1.21 0.34 0.64 4.25 0.27mTOR 0.192 0.692 14 10 10 30DNAPK 0.251 0.185 0.735 0.919 0.826 0.309PI4Kβ 2.78 4.02 3.7 7.39 100 4.09

Abl 0.075 0.063 0.052 0.024 1.5 0.026Hck 0.076 0.028 0.047 0.0044 0.436 0.01Src 0.025 0.01 0.033 0.005 0.286 0.031

Src (T/I) 4.81 4.01 2.1 1.65 12.6 0.729VEGFR 1.66 0.901 0.093 0.048 1.02 0.044EGFR 3.72 1.77 0.576 0.751 11 0.249EphB4 0.566 1.02 0.251 0.096 3.3 0.017

Me

OH

Me

OH

Me

OH

Me

OH

OH

F

OH

F

OH

BrOMe

BrOH

F

OH

F

Me

OH Me

O


R1

R2 Me

p110α 1.7 0.07 0.046 0.076 0.142 0.187p110β 4.6 0.173 0.24 0.235 0.32 0.613p110δ 0.206 0.023 0.027 0.041 0.112 0.065p110γ 7.9 0.039 0.104 0.044 0.054 0.209mTOR 1.1 0.548 0.045 0.127 0.156 0.037DNAPK 0.218 0.012 0.017 0.019 0.03 0.088PI4Kβ ND ND ND ND ND 2.5

Abl 0.319 0.109 0.021 0.023 0.045 0.014Hck 0.034 0.024 0.0041 0.0089 0.012 0.0092Src 0.072 0.061 0.01 0.0095 0.011 0.0069

Src (T/I) 7.44 100 1.9 1.9 1.69 0.865VEGFR 2.02 3 0.055 0.04 0.055 0.068EGFR 4.32 100 0.55 0.324 0.236 1.4EphB4 100 5.8 0.215 0.322 0.347 0.166


R1

R2

p110α 6.52 4.87 0.693 0.394 0.482 1.63p110β 6.47 16.3 0.481 0.169 1.58 0.364p110δ 3.03 4.35 1.37 0.716 0.777 0.213p110γ 0.808 0.606 0.502 0.448 1.07 0.633mTOR 11 2.9 2.2 0.833 1.5 30DNAPK 1.61 0.5 0.194 0.149 0.336 0.242PI4Kβ 31.9 12.4 ND ND ND 24

Abl 0.199 0.726 0.963 0.433 0.055 0.293Hck 0.161 1.4 0.258 0.071 0.011 0.042Src 0.101 0.405 0.09 0.018 0.024 0.017

Src (T/I) 100 100 100 100 100 100VEGFR 1.9 2.8 2.13 2.9 0.517 1.15EGFR 100 26 18 11 2.9 2.9EphB4 1 100 13 100 2 ND

NHNH2NH2CN

CN

F

OH

F

OH

F

OH

F

OHF

OH

F

OH

F

OH

Br

OO

Br

OH

Br

OH

Br

OH

Br

OH


R1

R2

p110α 0.525 0.347 5.76 9.15 0.65 0.66p110β 1.02 1.24 0.432 1.42 1.86 0.715p110δ 0.142 0.058 0.193 0.514 0.015 0.049p110γ 0.257 0.283 0.78 1.19 0.554 0.342mTOR 1.8 0.815 6.5 5 9.2 1DNAPK 0.11 0.107 0.543 0.61 0.151 0.277PI4Kβ 11.5 4.06 100 100 9.02 2.86

Abl 0.0043 0.0064 0.072 0.483 0.021 0.021Hck 0.0045 0.0057 0.025 0.086 0.021 0.051Src 0.00055 0.001 0.015 0.046 0.014 0.031

Src (T/I) 2.03 0.63 10.8 9.2 9.9 11VEGFR 0.106 0.077 0.369 1.75 0.401 0.555EGFR 0.318 0.366 8.7 15.6 4.8 2.6EphB4 0.064 0.054 0.492 100 0.882 0.611


R1

R2

p110α 0.244 5.25 1.099 100 0.543 0.135p110β 100 5.69 2.82 100 3.21 0.66p110δ 0.074 1.985 0.172 1.07 0.281 0.012p110γ 0.808 16.4 1.07 100 0.551 0.14mTOR 1.1 100 50 100 8.2 0.13DNAPK 0.37 2.74 0.049 3.69 0.824 0.017PI4Kβ ND 100 100 100 13 4.6

Abl 0.067 5.842 0.393 0.565 0.558 0.0055Hck 0.03 1.53 0.596 0.714 0.037 0.002Src 0.014 0.747 0.082 0.042 0.098 0.004

Src (T/I) 100 100 100 100 100 1.5VEGFR 0.178 11.8 100 100 100 0.02EGFR 0.775 100 100 100 3.86 0.158EphB4 0.254 100 18.7 100 0.244 0.106

ONH OMe NH

Cl

OH

F

OH

F

OH

F

OH

F

OH

F

OMe

F

OH

F

OH

F

OH

F

OH

F

OH

F

OH

NHN

NNMe

O

N


R1

R2

p110α 1.62 0.784 0.176 0.225 0.863 0.113p110β 3.62 3.53 1.35 0.941 0.864 0.55p110δ 1.62 1 0.15 0.103 0.203 0.02p110γ 0.637 0.08 0.293 0.147 0.366 0.058mTOR 2 0.88 0.23 0.439 2.4 0.377DNAPK 0.18 0.064 0.043 0.013 0.077 0.028PI4Kβ 14.6 12.9 ND 10 41.2 8.8

Abl 0.13 0.695 0.032 0.0012 0.505 0.021Hck 0.033 0.194 0.0058 0.00018 0.02 0.0088Src 0.073 0.284 0.011 0.00012 0.03 0.002

Src (T/I) 9.5 100 9.8 1.5 27 3.83VEGFR 0.554 1.14 0.062 0.0043 0.174 0.066EGFR 4.8 9.14 1.15 0.067 12.4 1.58EphB4 1 10 1.5 0.018 0.468 0.256


R1

R2

p110α 0.293 6.29 3.85 100 2.66 3.68p110β 0.809 6.98 0.962 100 2.7 2.64p110δ 0.012 5.47 0.207 6.05 0.199 0.156p110γ 0.043 1.82 1.39 12.5 1.53 2.41mTOR 0.45 15 4.8 3.2 3 3.6DNAPK 0.035 0.367 0.169 1.93 0.408 0.179PI4Kβ 100 100 100 100 100 100

Abl 0.017 1.044 0.082 7.93 4.5 1.28Hck 0.008 0.328 0.067 1.13 0.862 0.244Src 0.007 0.096 0.025 0.132 0.202 0.144

Src (T/I) 5.5 86.4 18.6 100 100 100VEGFR 0.103 3.12 0.435 7.16 3.78 1.28EGFR 1.4 9.38 2.01 2.72 100 20.5EphB4 0.519 8.05 0.352 100 7.27 3.71

O

ONHNH2 NH2

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

Cl

OH

NHHN

NHNHNH


R1

R2

p110α 0.357 0.086 0.092 ND 0.296 3.2p110β 0.744 0.742 1.45 1.28 2.53 100p110δ 0.187 0.088 0.161 ND 0.09 0.73p110γ 0.6 0.028 0.187 0.821 0.118 1.1mTOR 0.752 0.596 0.003 0.404 1.13 8DNAPK 0.022 0.009 0.036 0.873 0.048 1.26PI4Kβ ND ND 0.21 3.5 0.47 8.2

Abl 1.1 7.7 0.664 2.5 1.9 1.7Hck 0.708 7.2 0.026 0.113 0.076 0.18Src 0.408 2.9 0.043 0.476 0.054 0.16

Src (T/I) 33 4.3 0.424 17.2 2.1 13VEGFR 2.1 1.4 1.53 3.12 8.1 100EGFR 2.6 8.1 1.04 10.2 6.9 1.4EphB4 5 15 0.628 1.76 2.7 0.628


R1

R2 H Me

p110α 0.26 0.705 0.403 0.276 0.164 0.068p110β 2.1 1.36 0.216 0.809 0.697 0.261p110δ 0.061 0.198 0.076 0.047 0.074 0.01p110γ 0.19 0.523 0.2 0.355 0.34 0.12mTOR 1.2 0.71 0.098 0.02 0.003 0.04DNAPK 0.46 0.576 0.15 0.214 0.212 0.111PI4Kβ 0.87 3.4 100 100 3.9 100

Abl 1.5 0.784 0.53 0.177 0.09 0.056Hck 0.1 0.624 0.794 0.115 0.018 0.018Src 0.12 0.489 0.51 0.067 0.017 0.0063

Src (T/I) 3.6 12.7 ND 7.11 1.77 0.431VEGFR 100 1.06 ND 0.279 0.119 0.259EGFR 0.908 100 100 6.45 2.19 0.437EphB4 1.37 38.1 7.28 2.52 0.737 0.258

OMe

OH

OMe

OH

OMe

OH

OMe

OH

OMe

OH

OH

OMe

CN

OH

CN

OH

OH

FOH

FF

OMe

FF OMe


R1

R2

p110α 0.192 2.22 2.26 0.813 0.08 0.464p110β 0.949 2.67 2.27 3.18 0.23 0.525p110δ 0.013 0.215 0.68 0.255 0.048 0.197p110γ 0.113 0.46 1.27 0.381 0.2 1.08mTOR 0.17 5.1 3.9 0.11 3.1 100DNAPK 0.155 0.683 1.31 0.383 0.439 1.3PI4Kβ 7.8 15 18 5 5.9 ND

Abl 0.068 4 2.05 0.608 4.1 4.4Hck 0.01 1.2 0.311 0.082 1.1 0.44Src 0.004 0.241 0.131 0.135 0.53 0.293

Src (T/I) 0.633 100 100 4.96 15.2 100VEGFR 0.055 1.15 0.872 0.109 100 100EGFR 0.27 17 12.6 0.768 3.02 6.34EphB4 0.166 3.6 100 1.25 3.5 1.18


R1

R2 H

p110α 0.57 1.5 0.036 0.289 3.5 0.62p110β 5.4 3.8 0.27 0.232 4.82 1.67p110δ 0.12 0.067 0.008 0.057 0.473 0.042p110γ 1.9 2.3 0.15 0.087 0.909 5.16mTOR 1.7 1.2 1.7 2.5 100 3.3DNAPK 0.709 0.66 0.099 0.463 2.02 0.847PI4Kβ 9 ND ND 5.1 5.6 5.5

Abl 12 6.4 0.7 1 6.15 2.1Hck 0.92 2 0.27 0.117 6 0.551Src 3.9 3.2 0.36 0.18 100 0.361

Src (T/I) 5.9 16 6.3 2.6 100 100VEGFR 100 10 14.7 1.69 100 13.4EGFR 12 18 4.8 3.3 100 5.95EphB4 17 19 2.9 1.5 100 4.45

NHMe NH

OHMe

OHMe

OMe

OMe

OMe

OMe

OMe

OMe

OMe

OMe

OMe

OMe

OMe

OMeO

OMe

O

OMe

OMe

OH

OMe

OH

OMe

OH

O

OH


R1

R2

p110α 1.9 0.135 0.615 100 100 0.433p110β 2.45 0.294 9.86 100 100 0.492p110δ 0.276 0.017 0.177 1.9 37 0.103p110γ 0.311 0.116 0.353 100 100 0.723mTOR 4.8 6.5 0.616 100 5.5 2.4DNAPK 0.556 0.242 2.5 3.41 2.27 0.63PI4Kβ 12 1.5 100 3 3.6 4

Abl 14.6 0.85 6.05 0.83 11.2 6Hck 3.4 0.285 0.861 0.096 0.733 1.2Src 15.2 0.142 0.175 0.076 0.499 2

Src (T/I) 100 9.16 100 9.41 6.01 12.6VEGFR 100 2.07 100 100 5.29 100EGFR 5.08 1.4 1.16 5.14 3.66 54.2EphB4 100 0.456 100 0.76 1.1 14.7


R1

R2 H

p110α 1.37 1.91 1.25 100 3.74 1.5p110β 100 1.61 1.5 100 14.2 3.5p110δ 2.4 0.686 0.125 100 1.26 0.28p110γ 2.01 8.02 1.4 50 1.45 0.7mTOR 100 0.635 4.3 ND 3.6 100DNAPK 3.84 0.789 0.331 12 0.717 0.64PI4Kβ 25 4.9 7.4 ND 4.72 9.3

Abl 65 1.19 3.6 ND 0.208 0.26Hck 10.5 1.6 0.38 ND 0.046 0.037Src 9.8 1.43 0.38 ND 0.04 0.023

Src (T/I) 15.8 100 16 ND 7.81 2.9VEGFR 12.6 ND 4.36 ND 2.16 2.6EGFR 8.01 100 4.3 ND 2.12 3.85EphB4 100 1.09 2.98 ND 27.2 2.63

OH

O

OMe

O

OMe

O

OMe OMe

MeO OMeOMe

OMeO

OMe

O

OMe OMe

OHN

NOMe

OMeOH

NHOMe

NH


R1

R2 H H

p110α 10.4 3.4 0.65 0.52 14 17p110β >50 100 5.23 2.88 100 100p110δ 10.2 1.2 0.252 0.144 11 82p110γ 1.8 100 0.735 0.271 11 9.6mTOR 100 18 0.911 1.9 ND NDDNAPK 6.37 5.8 100 0.509 0.4 2.4PI4Kβ 29 ND 2.34 0.53 ND ND

Abl 0.0005 8.7 0.113 0.026 ND NDHck 0.0005 0.567 0.013 0.002 ND NDSrc 0.0005 0.341 0.011 0.0022 ND ND

Src (T/I) 0.025 28 2.03 1.32 ND NDVEGFR 2.23 0.194 0.566 0.752 ND NDEGFR 0.168 5.32 0.033 0.084 ND NDEphB4 100 11 0.764 0.045 ND ND


R1

R2 H H

p110α 100 12 3.1 2.9 10 4.6p110β 100 100 6.1 7.2 100p110δ 100 2.3 0.67 0.9 1.2 0.64p110γ 100 3 0.89 1.3 10 2.4mTOR ND ND 5.4 12 3.5 40DNAPK 100 5 1.35 0.883 2.68 1.23PI4Kβ ND ND 5.1 4.4 3.5 7.2

Abl ND ND 1.8 50 7.9 17Hck ND ND 2.4 5 0.51 50Src ND ND 0.53 3 0.89 5.9

Src (T/I) ND ND 12 100 12 50VEGFR ND ND 100 4.8 11 43EGFR ND ND 100 17 13 100EphB4 ND ND 100 13 100 10

OMe

NHBoc

OH

NHOMe

NH

OMe

HNMe

OHNH2

NO2NO2

NH2

OH CN CNCN


R1

R2

p110α 47 3 12.9 7.6 50 1.4p110β 100 100 6.1 50 100 3.3p110δ 6.3 1.4 6.9 1.9 100 0.41p110γ 3.5 6.2 ND 2.3 100 0.86mTOR 100 100 5.8 5.5 50 100DNAPK 6 2.6 4.28 2.75 10.1 1.96PI4Kβ ND ND 24 14 100 3.4

Abl 3.4 5 50 32 50 2Hck 1 4.3 13 9.2 13 1.4Src 1.5 4.4 12 8.7 50 2.1

Src (T/I) 100 100 100 100 100 100VEGFR 4.3 100 100 100 100 100EGFR 100 8.9 100 100 100 12EphB4 12 14 100 100 100 100


R1

R2

p110α 1.4 2.7 2.6 3.4 1.6 1.1p110β 5.9 7.2 7 8 ND 80p110δ 0.213 3.9 0.49 0.8 0.69 0.14p110γ 2.4 4.2 ND 1.2 0.94 1.4mTOR 2.4 30 10 16 0.766 0.934DNAPK 0.216 100 0.565 1.17 0.737 0.184PI4Kβ ND ND 5.5 9.1 2.8 1.7

Abl 0.688 1.3 50 16 4.5 2.6Hck 0.855 0.516 7.9 2.3 4.4 7.9Src 0.49 0.764 15 3.4 5.2 1.5

Src (T/I) 59 2.4 100 100 2.9 7VEGFR 1.2 1.4 100 100 1.02 1.05EGFR 9 10 100 2.8 5.96 9.9EphB4 15 7.1 3.1 3.8 17 23

F

CN

CN

F N

CNN

NN

O

O

F

O

F

F

OCl

ONH2

O H

O

SMe

HO


R1 I

R2

p110α 1.6 13.7 1 2.5 1.25 3p110β 2.1 5.2 1.9 5.8p110δ 0.65 1.4 0.21 0.24 0.25 0.68p110γ 0.68 7.2 0.9 4 2 0.99mTOR 5 3.8 4.3 5 6.5 0.08DNAPK 0.365 0.979 0.713 0.894 1.14 0.339PI4Kβ 4.9 2 4.9 2.7 6.4 5.7

Abl 13 11 11 58 25 3.2Hck 3.6 2.3 5.3 1.5 2.2 2.4Src 7.2 2.3 5 11 11 3.2

Src (T/I) 10 6.4 1.6 50 5.1 3.3VEGFR 100 5.89 4.6 98.8 4.98 1.68EGFR 100 12.8 100 100 17.8 0.579EphB4 100 100 18 15 15.9 2.91


R1

R2 H H H Me H H

p110α 100 12 100 10 17 100p110β 25 100 100 100 100 100p110δ 15 23 100 100 6.3 100p110γ 25 50 100 100 20 100mTOR ND ND ND ND ND NDDNAPK 100 5 100 10 0.67 100PI4Kβ ND ND ND ND ND ND

Abl ND ND ND ND ND NDHck ND ND ND ND ND NDSrc ND ND ND ND ND ND

Src (T/I) ND ND ND ND ND NDVEGFR ND ND ND ND ND NDEGFR ND ND ND ND ND NDEphB4 ND ND ND ND ND ND

O

Cl

ClOO

O OHNO

NHS O

Me O

HN S

O

MeO S

ONH2

O

SO

NH2

O

O

NH

SN

Supplementary Table 1 continuedName PP219 PP220 PP221 PP222 S2 PP223NCB # 219 220 221 222 8 223

R1

R2 Me H H H Me Me

p110α 100 100 100 3 1.1 100p110β 100 100 100 100 16 100p110δ 100 100 100 0.81 0.8 100p110γ 100 100 50 0.97 0.57 100mTOR ND ND ND ND 100 NDDNAPK 0.86 100 4.4 0.15 0.029 0.42PI4Kβ ND ND ND ND ND ND

Abl ND ND ND ND 1.2 NDHck ND ND ND ND 0.26 NDSrc ND ND ND ND 1.1 ND

Src (T/I) ND ND ND ND 7.2 NDVEGFR ND ND ND ND 1.1 NDEGFR ND ND ND ND 0.388 NDEphB4 ND ND ND ND 4.3 ND


R1

R2

p110α ND 5.6 4.8 100 12 100p110β ND 50 32 100 50 50p110δ 0.75 2.9 1.2 100 6.7 28p110γ ND 3.6 4.5 100 6.8 17mTOR ND ND ND ND ND NDDNAPK 0.21 0.35 0.2 0.42 0.78 2.1PI4Kβ ND ND ND ND ND ND




R1

R2

p110α 33 9.3 5 29 ND NDp110β 50 100 100 100 ND NDp110δ 21 3 1.8 12 3.6 3p110γ 6.3 5.1 4 16 ND NDmTOR ND ND ND ND ND NDDNAPK 0.34 0.63 0.4 1.1 1.1 0.28PI4Kβ ND ND ND ND ND ND




R1

R2 Me Me

p110α 100 1.3 15 2.8 1.8 0.035p110β 5.3 3.2 100 16 2.7 1.4p110δ 3.7 0.059 1.8 0.4 0.453 0.09p110γ 2.7 0.587 100 2.7 1.3 0.22mTOR 100 0.109 2.2 1.9 0.243 3DNAPK 0.26 0.1 1.1 0.317 0.214 0.036PI4Kβ ND 7.4 ND ND ND ND

Abl 15 0.057 2 1 0.759 100Hck 0.54 0.02 1.6 0.422 0.326 100Src 2.4 0.055 2.1 1.53 0.716 12

Src (T/I) ND 0.055 3.4 5.8 17 3VEGFR 0.55 0.01 100 5.6 1.6 12EGFR 21 0.088 100 1 0.54 34EphB4 1.1 0.095 2.5 1.7 0.66 10

N NN

NH2

O

OHOEtN

Cl


R1

R2

p110α 0.0175 0.07 0.462 0.8 0.206 1.7p110β 0.244 2.7 1.6 5.5 0.975 6.5p110δ 0.006 0.089 0.056 0.123 0.049 0.409p110γ 0.043 0.59 0.461 0.184 0.095 100mTOR 0.14 0.55 0.565 0.113 0.632 6.8DNAPK 0.008 0.047 0.102 0.08 0.126 8.1PI4Kβ 0.049 ND ND ND ND ND

Abl 0.134 1.5 5.1 3.8 2.2 2Hck 0.088 0.54 2.2 0.878 0.946 12Src 0.235 0.81 100 2.7 1 30

Src (T/I) 3.6 7.4 9.6 5.7 100 100VEGFR 0.31 4.4 1.1 0.96 13 12EGFR 0.61 2.1 100 100 18 70EphB4 0.23 2.7 2.7 3.2 32 100


R1

R2

p110α 0.08 100 100 0.245 0.467 2.8p110β 0.467 15 100 0.5 4.6 8.9p110δ 0.066 100 100 0.375 1 1.5p110γ 0.089 100 100 0.242 0.586 7.3mTOR 0.02 8.7 100 0.033 0.783 3.2DNAPK 0.0005 18 100 0.014 0.314 1.9PI4Kβ ND ND ND ND ND ND

Abl 0.328 35 100 2 3 65Hck 0.248 2.7 1.1 17 4.3 17Src 0.131 7.2 100 7.6 0.8 12

Src (T/I) 3.8 9.4 100 100 100 100VEGFR 8.1 100 100 5.1 50 100EGFR 7.4 6.7 1.4 12 7 100EphB4 14 12 100 18 87 17

N N

NHN

O

N

N

HNO

HNO

NMe

NCl

NN

N

NNH2

NNHNH2 N

NHBoc


R1

R2 Me

p110α 5.1 6.8 2.4 0.344 32 0.443p110β 12 8.1 25 1.1 100 1.3p110δ 5.2 3.9 1.7 0.12 3.4 0.085p110γ 6.9 3 3 0.06 100 0.167mTOR 10 100 1.3 0.426 2.2 1.5DNAPK 2 0.283 0.26 0.046 2.7 0.049PI4Kβ ND ND ND ND ND ND

Abl 6.4 100 2.2 2 12 2.1Hck 3.4 17 2.5 1.1 100 1.8Src 2.7 15 1.6 0.525 0.8 11

Src (T/I) 100 100 8.7 100 100 100VEGFR 7 100 100 14 49 19EGFR 100 10 29 100 25 100EphB4 14 100 8.6 18 12 12


R1

R2 Me

p110α 0.752 0.091 0.12 0.052 0.652 0.531p110β 0.476 1.6 1 1.4 3.22 4.1p110δ 0.11 0.14 0.077 0.15 0.173 0.228p110γ 0.228 0.64 1.3 1.1 0.123 0.435mTOR 1.1 0.412 0.06 0.013 0.364 0.407DNAPK 0.359 0.12 0.071 0.06 0.09 0.08PI4Kβ ND ND ND ND 4.1 9.8

Abl 4.3 0.31 0.022 0.018 0.295 0.234Hck 16 0.42 0.01 0.008 0.015 0.0054Src 0.55 0.44 0.02 0.014 0.039 0.008

Src (T/I) 100 6.8 0.48 0.22 0.103 0.206VEGFR 100 0.94 0.019 0.012 0.076 0.009EGFR 18 15 0.38 0.26 0.176 0.734EphB4 3.8 2.9 0.34 0.19 0.23 0.105

N N

NMe

N

NH2

O

O

O

O

O

O

N

HN

N

HN

N

HN

HN

HN

Supplementary Table 1 continuedName PP135 PP136 PP137 PP138 PP160NCB # 135 136 137 138 160

R1

R2

p110α 0.642 0.831 0.145 0.209 1.6p110β 0.854 1.1 0.55 1.5 16p110δ 0.037 0.443 0.1 0.142 0.276p110γ 0.627 31 0.113 0.09 0.451mTOR 0.457 0.205 0.657 1.1 1.8DNAPK 0.349 0.244 0.105 0.085 0.023PI4Kβ ND ND ND ND ND

Abl 0.69 0.816 0.498 0.469 0.327Hck 0.153 0.148 0.053 0.033 0.035Src 0.157 0.211 0.073 0.049 0.058

Src (T/I) 2.4 1.3 0.393 0.261 2.2VEGFR 0.715 0.172 0.295 0.411 6.3EGFR 0.955 0.539 1.1 0.571 0.207EphB4 100 2.2 2.3 2 1.3


R1

R2 Me

p110α 0.193 0.069 0.082 0.144 1.8 2.6p110β 0.658 1 1.9 0.548 2.9 5p110δ 0.068 0.12 0.19 0.162 0.66 0.34p110γ 0.101 0.33 0.83 0.625 1.8 2mTOR 1.64 1.2 0.596 7 8.3 0.467DNAPK 0.01 0.077 0.11 3.4 0.15 0.088PI4Kβ ND ND ND ND ND ND

Abl 0.52 0.11 0.14 0.087 1.5 0.2Hck 0.4 0.03 0.037 0.076 0.22 0.018Src 0.088 0.069 0.042 0.103 0.25 0.042

Src (T/I) 22 4.9 0.93 2.3 11 1.2VEGFR 6.7 0.42 0.33 0.215 11 0.47EGFR 9.6 2.5 0.76 1.2 2.9 0.22EphB4 21 1.6 1.4 4.5 5 0.29

NHN

H2N

NHN

NHN

NHNF

NH NH

NHN N

HN

NHN

H2NN

MeN

Me


R1

R2

p110α 4 100 6.3 1.96 0.45 9.85p110β 7 52 15 2.2 ND 4.75p110δ 0.966 72 2.3 0.102 0.125 2.02p110γ 1.5 100 2.4 1.27 0.14 1.022mTOR 0.493 7.6 4.8 0.008 0.22 8DNAPK 0.054 0.222 0.053 0.408 0.019 0.953PI4Kβ ND ND ND 22 4.2 2.6

Abl 0.165 14 13 3.6 12 2.6Hck 0.005 100 8.9 1.18 0.27 1.2Src 0.017 23 11 1.4 1.2 0.037

Src (T/I) 0.25 100 100 5.14 11.7 1VEGFR 0.134 100 100 1.54 2.2 6.9EGFR 0.085 45 12 4.38 0.638 4.6EphB4 0.17 100 21 3.46 2.05 2.9


R1

R2

p110α 3.04 2.56 100 1.06 2.69 3.4p110β 10.6 45 100 67.2 28.7 0.44p110δ 0.446 2.48 1.03 0.436 0.429 3.7p110γ 1.14 0.799 0.873 0.347 0.239 5.9mTOR 5.4 0.3 0.576 3.4 1.6 100DNAPK 0.929 0.43 0.916 0.303 0.22 1.2PI4Kβ 3.5 13 100 5.7 8 14

Abl 2.2 0.527 0.92 0.538 0.893 13Hck 0.057 0.009 0.048 0.091 0.056 1.3Src 0.057 0.046 0.194 0.238 0.14 3

Src (T/I) 1.3 0.116 0.44 12 15 50VEGFR 4.13 0.153 0.84 2.9 1.22 100EGFR 1.4 0.179 0.391 0.964 0.575 8.3EphB4 1.6 0.353 0.871 0.675 0.38 100

NH

OHCN NN N N

OONONBoc

NBocN

N

NH

NH

OH

Supplementary Table 1 continuedName PP156 PP95 PP233 PP54 PP234 PP42NCB # 156 95 PP233 54 234 42

R1

R2 H Me

p110α 2.2 1.95 4.9 2.2 1.3 3.4p110β 9.2 46.8 30 1.4 15 50p110δ 2 0.643 7.4 1.3 1.2 1.9p110γ 22 0.43 4 1.1 1.6 2.4mTOR 3.6 4.5 ND 1.9 ND 4.5DNAPK 0.186 0.546 0.88 0.489 0.085 0.288PI4Kβ ND 5 ND 9.1 ND 12

Abl 8.4 5.2 ND 1.3 ND 50Hck 4.8 0.889 ND 1 ND 12Src 3.5 1.03 ND 0.93 ND 12

Src (T/I) 52 100 ND 6.9 ND 50VEGFR 13 100 ND 21 ND 1.64EGFR 0.937 5 ND 15 ND 100EphB4 17 3 ND 6.76 ND 22

Name Gefitinib Dasatinib Sorafenib SunitinibNCB # 18 11 17 12

p110α >50 38 >50 11p110β >50 >50 >50 >50p110δ >50 30 >50 12p110γ >50 >50 >50 13mTOR >50 35 >50 >50DNAPK >50 >50 >50 >50

Abl 1.2 <0.001 0.12 0.41Hck 0.11 <0.001 0.53 0.19Src 1.1 <0.001 0.39 0.4

Src (T338I) 1.5 3 0.78 0.11VEGFR2 1.9 >50 0.027 0.095

EGFR <0.001 0.02 >50 18EphB4 1 <0.001 1.9 >50

cKI >50 <0.001 0.25 <0.001PDGFR 0.19 <0.001 <0.001 <0.001

Ret 1.7 2.1 0.009 <0.001InsR >50 >50 5.3 3.2

O

O

O

NH

HN O

SS

CHO

O

O

Supplementary Table 1. IC50 values for pyrazolopyrimidines and clinically approved tyrosine kinase inhibitors against tyrosine kinases and PI3-Ks. All IC50 values were measured against purified kinases in the presence of 10 μM ATP.

Supplementary Table 2 Percent Inhibition

Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP102 10 ABL1 81 83 82PP102 10 ABL1 E255K 79 80 80PP102 10 ABL1 G250E 66 69 67PP102 10 ABL1 T315I 16 17 17PP102 10 ABL1 Y253F 83 85 84PP102 10 ABL2 (Arg) 69 70 70PP102 10 ACVR1B (ALK4) 98 100 99PP102 10 ADRBK1 (GRK2) 5 2 4PP102 10 ADRBK2 (GRK3) -4 3 0PP102 10 AKT1 (PKB alpha) 6 7 7PP102 10 AKT2 (PKB beta) 2 5 3PP102 10 AKT3 (PKB gamma) 4 15 9PP102 10 ALK 10 3 7PP102 10 AMPK A1/B1/G1 (PRKA A1/B1/G1) 7 10 8PP102 10 AURKB (Aurora B) 1 3 2PP102 10 AURKC (Aurora C) -3 5 1PP102 10 BLK 68 68 68PP102 10 BMX 78 78 78PP102 100 BRAF 55 66 60PP102 100 BRAF V599E 78 81 80PP102 10 BRSK1 (SAD1) 7 5 6PP102 10 BTK 57 57 57PP102 10 CAMK1D (CaMKI delta) 2 6 4PP102 10 CAMK2A (CaMKII alpha) -7 0 -3PP102 10 CAMK2B (CaMKII beta) 4 4 4PP102 10 CAMK2D (CaMKII delta) 5 5 5PP102 10 CAMK4 (CaMKIV) -6 2 -2PP102 10 CDC42 BPA (MRCKA) 8 -7 0PP102 10 CDC42 BPB (MRCKB) 2 -22 -10PP102 10 CDK1/cyclin B 6 6 6PP102 10 CDK2/cyclin A -2 6 2PP102 10 CDK5/p35 -3 3 0PP102 10 CHEK1 (CHK1) -2 2 0PP102 100 CLK1 1 2 2PP102 10 CLK2 8 11 10PP102 100 CLK3 8 7 8PP102 10 CSF1R (FMS) 36 37 37PP102 10 CSK 32 33 32PP102 10 CSNK1A1 (CK1 alpha 1) 26 30 28PP102 10 CSNK1D (CK1 delta) 87 87 87PP102 10 CSNK1E (CK1 epsilon) 97 100 98PP102 10 CSNK1G1 (CK1 gamma 1) -5 1 -2PP102 10 CSNK1G2 (CK1 gamma 2) -5 -10 -7PP102 10 CSNK1G3 (CK1 gamma 3) -4 -14 -9PP102 10 CSNK2A1 (CK2 alpha 1) 19 -1 9PP102 10 CSNK2A2 (CK2 alpha 2) 9 8 9PP102 10 DAPK3 (ZIPK) 0 -1 0PP102 100 DCAMKL2 (DCK2) -1 0 0PP102 10 DYRK1A 9 13 11

Supplementary Table 2 continued Percent Inhibition

Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP102 10 DYRK1B 9 12 10PP102 10 DYRK3 13 0 6PP102 10 DYRK4 0 1 0PP102 10 EGFR (ErbB1) 38 34 36PP102 10 EGFR L858R (ErbB1 L858R) 58 57 58PP102 10 EGFR L861Q (ErbB1 L861Q) 37 39 38PP102 10 EPHA1 90 91 90PP102 10 EPHA2 82 74 78PP102 10 EPHA3 28 30 29PP102 100 EPHA4 67 69 68PP102 10 EPHA5 81 81 81PP102 10 EPHA8 94 92 93PP102 10 EPHB1 85 83 84PP102 10 EPHB2 90 91 91PP102 10 EPHB3 51 51 51PP102 10 EPHB4 80 80 80PP102 10 ERBB2 (HER2) 32 35 33PP102 10 ERBB4 (HER4) 28 29 29PP102 10 FER 12 13 13PP102 10 FES (FPS) 22 22 22PP102 10 FGFR1 30 32 31PP102 10 FGFR2 26 31 28PP102 10 FGFR3 29 29 29PP102 10 FGFR3 K650E 14 19 16PP102 10 FGFR4 25 28 26PP102 10 FGR 90 89 90PP102 100 FLT1 (VEGFR1) 16 18 17PP102 10 FLT3 24 29 26PP102 10 FLT3 D835Y 14 14 14PP102 10 FLT4 (VEGFR3) 0 0 0PP102 10 FRK (PTK5) 74 75 74PP102 10 FYN 83 85 84PP102 10 GRK4 -3 1 -1PP102 10 GRK5 0 -12 -6PP102 10 GRK6 0 1 1PP102 10 GRK7 8 11 10PP102 10 GSK3A (GSK3 alpha) 9 10 9PP102 10 GSK3B (GSK3 beta) 5 5 5PP102 10 HCK 82 83 83PP102 10 HIPK1 (Myak) 5 2 3PP102 10 HIPK4 13 14 14PP102 10 IGF1R -1 4 2PP102 10 IKBKB (IKK beta) 1 -1 0PP102 10 INSR -1 0 -1PP102 100 INSRR (IRR) 2 0 1PP102 10 IRAK4 0 0 0PP102 10 ITK -1 1 0PP102 10 JAK2 0 6 3PP102 100 JAK2 JH1 JH2 -7 -5 -6


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP102 10 JAK2 JH1 JH2 V617F 1 6 4PP102 10 JAK3 -1 1 0PP102 10 KDR (VEGFR2) 82 85 83PP102 100 KIT 23 21 22PP102 10 KIT T670I 10 17 13PP102 10 LCK 84 87 85PP102 10 LTK (TYK1) 4 9 6PP102 10 LYN A 85 86 85PP102 10 LYN B 86 87 86PP102 100 MAP2K1 (MEK1) 16 18 17PP102 100 MAP2K2 (MEK2) 23 25 24PP102 100 MAP2K6 (MKK6) 6 4 5PP102 100 MAP3K8 (COT) 12 15 14PP102 100 MAP3K9 (MLK1) 4 4 4PP102 10 MAP4K2 (GCK) 21 25 23PP102 10 MAP4K4 (HGK) 83 86 85PP102 10 MAP4K5 (KHS1) 42 49 45PP102 10 MAPK1 (ERK2) 17 19 18PP102 100 MAPK11 (p38 beta) 11 11 11PP102 10 MAPK12 (p38 gamma) 5 5 5PP102 10 MAPK13 (p38 delta) 7 8 7PP102 100 MAPK14 (p38 alpha) 6 7 6PP102 10 MAPK3 (ERK1) 11 12 12PP102 10 MAPKAPK2 0 -2 -1PP102 10 MAPKAPK3 -2 -1 -1PP102 10 MAPKAPK5 (PRAK) 6 4 5PP102 10 MARK1 (MARK) 3 2 2PP102 10 MARK2 2 0 1PP102 10 MATK (HYL) 4 0 2PP102 10 MERTK (cMER) 19 23 21PP102 10 MET (cMet) 1 7 4PP102 10 MET M1250T 0 -1 0PP102 10 MINK1 83 83 83PP102 10 MST1R (RON) 40 44 42PP102 100 MST4 -16 -21 -19PP102 100 MUSK 3 -1 1PP102 100 MYLK2 (skMLCK) 4 3 4PP102 10 NEK1 39 48 44PP102 10 NEK2 25 30 27PP102 100 NEK4 4 3 4PP102 10 NEK6 2 2 2PP102 100 NEK7 0 3 2PP102 10 NEK9 4 6 5PP102 100 NTRK1 (TRKA) 32 37 35PP102 10 NTRK2 (TRKB) 21 22 21PP102 10 NTRK3 (TRKC) 36 36 36PP102 10 PAK2 (PAK65) 4 7 5PP102 10 PAK3 23 20 21PP102 10 PAK4 12 8 10


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP102 10 PAK6 2 6 4PP102 10 PAK7 (KIAA1264) 3 6 4PP102 100 PASK 4 1 3PP102 10 PDGFRA (PDGFR alpha) 39 40 40PP102 10 PDGFRA D842V 31 30 30PP102 100 PDGFRA T674I 14 10 12PP102 10 PDGFRB (PDGFR beta) 32 35 33PP102 100 PDK1 5 8 7PP102 10 PHKG1 -6 6 0PP102 10 PHKG2 -20 2 -9PP102 10 PIM1 6 6 6PP102 10 PIM2 2 4 3PP102 10 PKN1 (PRK1) -3 8 2PP102 10 PLK1 0 2 1PP102 10 PLK2 6 12 9PP102 10 PLK3 12 10 11PP102 10 PRKACA (PKA) 32 33 33PP102 10 PRKCA (PKC alpha) 36 44 40PP102 10 PRKCB1 (PKC beta I) 24 26 25PP102 10 PRKCB2 (PKC beta II) 14 11 13PP102 10 PRKCD (PKC delta) 9 14 12PP102 10 PRKCE (PKC epsilon) 2 19 10PP102 10 PRKCG (PKC gamma) 44 40 42PP102 10 PRKCH (PKC eta) 3 2 2PP102 10 PRKCI (PKC iota) 2 2 2PP102 10 PRKCN (PKD3) 70 73 72PP102 10 PRKCQ (PKC theta) 5 3 4PP102 10 PRKCZ (PKC zeta) 9 17 13PP102 10 PRKD1 (PKC mu) 69 72 70PP102 10 PRKD2 (PKD2) 79 82 81PP102 10 PRKG1 13 14 14PP102 10 PRKG2 (PKG2) -4 0 -2PP102 10 PRKX 13 13 13PP102 10 PTK2B (FAK2) 4 6 5PP102 100 PTK6 (Brk) 88 89 89PP102 100 RAF1 (cRAF) 69 74 72PP102 10 RET 94 92 93PP102 10 RET V804L 4 5 4PP102 10 RET Y791F 93 93 93PP102 10 ROCK1 14 4 9PP102 10 ROCK2 1 6 4PP102 100 ROS1 6 4 5PP102 10 RPS6KA1 (RSK1) 3 3 3PP102 10 RPS6KA2 (RSK3) 7 14 11PP102 10 RPS6KA3 (RSK2) 1 4 2PP102 10 RPS6KA4 (MSK2) 1 5 3PP102 10 RPS6KA5 (MSK1) 1 2 2PP102 10 RPS6KA6 (RSK4) 10 13 11PP102 10 RPS6KB1 (p70S6K) -1 5 2


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP102 10 SGK (SGK1) -1 4 1PP102 10 SGK2 2 2 2PP102 10 SGKL (SGK3) -2 1 0PP102 10 SRC 61 59 60PP102 10 SRC N1 66 66 66PP102 100 SRMS (Srm) 61 64 63PP102 10 SRPK1 4 6 5PP102 10 SRPK2 -1 7 3PP102 10 STK22B (TSSK2) 4 2 3PP102 10 STK22D (TSSK1) 3 4 3PP102 100 STK23 (MSSK1) 0 1 1PP102 100 STK24 (MST3) 10 -1 4PP102 10 STK25 (YSK1) -4 -8 -6PP102 10 STK3 (MST2) 11 19 15PP102 10 STK4 (MST1) 0 9 4PP102 10 STK6 (Aurora A) 0 3 2PP102 10 SYK 1 7 4PP102 100 TAOK2 (TAO1) 2 9 6PP102 10 TBK1 4 5 5PP102 10 TEK (Tie2) 12 12 12PP102 10 TYRO3 (RSE) 17 18 17PP102 10 YES1 89 88 88PP102 10 ZAP70 15 9 12

PP121 10 ABL1 96 97 96PP121 10 ABL1 E255K 97 98 98PP121 10 ABL1 G250E 96 97 96PP121 10 ABL1 T315I 36 40 38PP121 10 ABL1 Y253F 97 95 96PP121 10 ABL2 (Arg) 97 96 97PP121 10 ACVR1B (ALK4) 92 89 91PP121 10 ADRBK1 (GRK2) 5 6 6PP121 10 ADRBK2 (GRK3) 6 6 6PP121 10 AKT1 (PKB alpha) 9 8 9PP121 10 AKT2 (PKB beta) 1 5 3PP121 10 AKT3 (PKB gamma) 38 40 39PP121 10 ALK 72 76 74PP121 10 AMPK A1/B1/G1 (PRKA A1/B1/G1) 8 6 7PP121 10 AURKB (Aurora B) 1 5 3PP121 10 AURKC (Aurora C) 9 7 8PP121 10 BLK 95 96 96PP121 10 BMX 84 86 85PP121 100 BRAF 95 99 97PP121 100 BRAF V599E 94 95 94PP121 10 BRSK1 (SAD1) 5 8 7PP121 10 BTK 82 86 84PP121 10 CAMK1D (CaMKI delta) 5 9 7PP121 10 CAMK2A (CaMKII alpha) -3 -3 -3PP121 10 CAMK2B (CaMKII beta) 5 4 5


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP121 10 CAMK2D (CaMKII delta) 3 7 5PP121 10 CAMK4 (CaMKIV) 2 7 5PP121 10 CDC42 BPA (MRCKA) -11 4 -4PP121 10 CDC42 BPB (MRCKB) -17 1 -8PP121 10 CDK1/cyclin B 5 4 4PP121 10 CDK2/cyclin A 13 8 11PP121 10 CDK5/p35 10 0 5PP121 10 CHEK1 (CHK1) -1 -1 -1PP121 100 CLK1 2 2 2PP121 10 CLK2 43 47 45PP121 100 CLK3 5 3 4PP121 10 CSF1R (FMS) 83 86 84PP121 10 CSK 38 40 39PP121 10 CSNK1A1 (CK1 alpha 1) 46 46 46PP121 10 CSNK1D (CK1 delta) 81 79 80PP121 10 CSNK1E (CK1 epsilon) 94 92 93PP121 10 CSNK1G1 (CK1 gamma 1) 1 -1 0PP121 10 CSNK1G2 (CK1 gamma 2) -1 3 1PP121 10 CSNK1G3 (CK1 gamma 3) -2 1 0PP121 10 CSNK2A1 (CK2 alpha 1) -1 3 1PP121 10 CSNK2A2 (CK2 alpha 2) 5 9 7PP121 10 DAPK3 (ZIPK) 0 0 0PP121 100 DCAMKL2 (DCK2) 2 2 2PP121 10 DYRK1A 8 9 9PP121 10 DYRK1B 6 7 7PP121 10 DYRK3 -2 -3 -2PP121 10 DYRK4 -1 1 0PP121 10 EGFR (ErbB1) 73 69 71PP121 10 EGFR L858R (ErbB1 L858R) 85 88 87PP121 10 EGFR L861Q (ErbB1 L861Q) 76 81 78PP121 10 EPHA1 91 92 91PP121 10 EPHA2 71 82 77PP121 10 EPHA3 53 57 55PP121 100 EPHA4 67 72 69PP121 10 EPHA5 81 83 82PP121 10 EPHA8 89 89 89PP121 10 EPHB1 87 85 86PP121 10 EPHB2 85 87 86PP121 10 EPHB3 77 79 78PP121 10 EPHB4 85 87 86PP121 10 ERBB2 (HER2) 18 19 18PP121 10 ERBB4 (HER4) 64 64 64PP121 10 FER 16 17 16PP121 10 FES (FPS) 37 36 37PP121 10 FGFR1 96 95 95PP121 10 FGFR2 92 91 91PP121 10 FGFR3 89 93 91PP121 10 FGFR3 K650E 83 87 85PP121 10 FGFR4 80 79 80


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP121 10 FGR 100 100 100PP121 100 FLT1 (VEGFR1) 77 73 75PP121 10 FLT3 28 31 29PP121 10 FLT3 D835Y 22 24 23PP121 10 FLT4 (VEGFR3) 1 1 1PP121 10 FRK (PTK5) 98 98 98PP121 10 FYN 99 98 98PP121 10 GRK4 0 -5 -2PP121 10 GRK5 -3 -1 -2PP121 10 GRK6 3 2 2PP121 10 GRK7 19 22 20PP121 10 GSK3A (GSK3 alpha) 23 22 23PP121 10 GSK3B (GSK3 beta) 10 10 10PP121 10 HCK 96 97 97PP121 10 HIPK1 (Myak) 2 3 2PP121 10 HIPK4 10 9 9PP121 10 IGF1R 55 59 57PP121 10 IKBKB (IKK beta) -1 2 1PP121 10 INSR 31 22 27PP121 100 INSRR (IRR) 12 18 15PP121 10 IRAK4 -1 4 2PP121 10 ITK 31 30 31PP121 10 JAK2 10 7 9PP121 100 JAK2 JH1 JH2 25 23 24PP121 10 JAK2 JH1 JH2 V617F 58 62 60PP121 10 JAK3 2 0 1PP121 10 KDR (VEGFR2) 99 97 98PP121 100 KIT 37 36 36PP121 10 KIT T670I 8 9 9PP121 10 LCK 97 97 97PP121 10 LTK (TYK1) 54 56 55PP121 10 LYN A 97 97 97PP121 10 LYN B 98 98 98PP121 100 MAP2K1 (MEK1) 4 7 5PP121 100 MAP2K2 (MEK2) 5 7 6PP121 100 MAP2K6 (MKK6) 6 10 8PP121 100 MAP3K8 (COT) 10 14 12PP121 100 MAP3K9 (MLK1) 12 17 14PP121 10 MAP4K2 (GCK) 91 91 91PP121 10 MAP4K4 (HGK) 39 36 38PP121 10 MAP4K5 (KHS1) 52 60 56PP121 10 MAPK1 (ERK2) 10 10 10PP121 100 MAPK11 (p38 beta) 7 9 8PP121 10 MAPK12 (p38 gamma) 2 3 3PP121 10 MAPK13 (p38 delta) 4 5 4PP121 100 MAPK14 (p38 alpha) 1 2 1PP121 10 MAPK3 (ERK1) 6 7 6PP121 10 MAPKAPK2 -3 -3 -3PP121 10 MAPKAPK3 0 -4 -2


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP121 10 MAPKAPK5 (PRAK) 4 2 3PP121 10 MARK1 (MARK) 22 25 24PP121 10 MARK2 24 32 28PP121 10 MATK (HYL) -5 1 -2PP121 10 MERTK (cMER) 13 11 12PP121 10 MET (cMet) 1 3 2PP121 10 MET M1250T -5 -5 -5PP121 10 MINK1 31 30 30PP121 10 MST1R (RON) -11 -17 -14PP121 100 MST4 62 64 63PP121 100 MUSK 3 2 3PP121 100 MYLK2 (skMLCK) 4 0 2PP121 10 NEK1 42 48 45PP121 10 NEK2 13 11 12PP121 100 NEK4 5 5 5PP121 10 NEK6 1 2 1PP121 100 NEK7 3 -2 0PP121 10 NEK9 4 7 6PP121 100 NTRK1 (TRKA) 30 32 31PP121 10 NTRK2 (TRKB) 11 13 12PP121 10 NTRK3 (TRKC) 29 29 29PP121 10 PAK2 (PAK65) 7 -1 3PP121 10 PAK3 15 17 16PP121 10 PAK4 24 29 26PP121 10 PAK6 16 12 14PP121 10 PAK7 (KIAA1264) 24 23 23PP121 100 PASK 2 2 2PP121 10 PDGFRA (PDGFR alpha) 66 68 67PP121 10 PDGFRA D842V 79 87 83PP121 100 PDGFRA T674I -1 5 2PP121 10 PDGFRB (PDGFR beta) 73 71 72PP121 100 PDK1 3 2 2PP121 10 PHKG1 7 1 4PP121 10 PHKG2 -2 -14 -8PP121 10 PIM1 7 7 7PP121 10 PIM2 2 3 3PP121 10 PKN1 (PRK1) 7 4 5PP121 10 PLK1 16 16 16PP121 10 PLK2 3 1 2PP121 10 PLK3 0 11 6PP121 10 PRKACA (PKA) 5 7 6PP121 10 PRKCA (PKC alpha) 40 38 39PP121 10 PRKCB1 (PKC beta I) 42 38 40PP121 10 PRKCB2 (PKC beta II) 35 37 36PP121 10 PRKCD (PKC delta) 25 17 21PP121 10 PRKCE (PKC epsilon) 73 74 73PP121 10 PRKCG (PKC gamma) 56 59 58PP121 10 PRKCH (PKC eta) 51 49 50PP121 10 PRKCI (PKC iota) 28 22 25


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP121 10 PRKCN (PKD3) 83 80 82PP121 10 PRKCQ (PKC theta) 34 39 37PP121 10 PRKCZ (PKC zeta) 22 15 18PP121 10 PRKD1 (PKC mu) 74 72 73PP121 10 PRKD2 (PKD2) 89 89 89PP121 10 PRKG1 13 16 14PP121 10 PRKG2 (PKG2) 8 11 9PP121 10 PRKX 14 13 13PP121 10 PTK2B (FAK2) 29 33 31PP121 100 PTK6 (Brk) 97 99 98PP121 100 RAF1 (cRAF) 84 86 85PP121 10 RET 100 101 100PP121 10 RET V804L 10 12 11PP121 10 RET Y791F 99 99 99PP121 10 ROCK1 22 34 28PP121 10 ROCK2 22 19 21PP121 100 ROS1 7 12 9PP121 10 RPS6KA1 (RSK1) 0 2 1PP121 10 RPS6KA2 (RSK3) 10 24 17PP121 10 RPS6KA3 (RSK2) 12 8 10PP121 10 RPS6KA4 (MSK2) 10 10 10PP121 10 RPS6KA5 (MSK1) 5 3 4PP121 10 RPS6KA6 (RSK4) 9 7 8PP121 10 RPS6KB1 (p70S6K) 2 3 3PP121 10 SGK (SGK1) 1 3 2PP121 10 SGK2 2 2 2PP121 10 SGKL (SGK3) 1 0 0PP121 10 SRC 95 95 95PP121 10 SRC N1 99 99 99PP121 100 SRMS (Srm) 79 77 78PP121 10 SRPK1 3 4 3PP121 10 SRPK2 7 5 6PP121 10 STK22B (TSSK2) 0 0 0PP121 10 STK22D (TSSK1) 9 9 9PP121 100 STK23 (MSSK1) -1 2 1PP121 100 STK24 (MST3) 70 74 72PP121 10 STK25 (YSK1) 102 103 102PP121 10 STK3 (MST2) 83 82 83PP121 10 STK4 (MST1) 82 84 83PP121 10 STK6 (Aurora A) 12 11 11PP121 10 SYK 21 20 20PP121 100 TAOK2 (TAO1) 12 7 10PP121 10 TBK1 2 2 2PP121 10 TEK (Tie2) 2 11 7PP121 10 TYRO3 (RSE) 37 42 40PP121 10 YES1 98 99 98PP121 10 ZAP70 24 19 22

PP242 10 ABL1 44 35 40


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP242 10 ABL1 E255K 22 20 21PP242 10 ABL1 G250E 16 13 14PP242 10 ABL1 T315I 26 18 22PP242 10 ABL1 Y253F 42 42 42PP242 10 ABL2 (Arg) 23 22 22PP242 10 ACVR1B (ALK4) 17 20 18PP242 10 ADRBK1 (GRK2) 9 10 10PP242 10 ADRBK2 (GRK3) 13 8 10PP242 10 AKT1 (PKB alpha) 3 7 5PP242 10 AKT2 (PKB beta) 7 4 6PP242 10 AKT3 (PKB gamma) 9 11 10PP242 10 ALK 30 32 31PP242 10 AMPK A1/B1/G1 (PRKA A1/B1/G1) 21 21 21PP242 10 AURKB (Aurora B) 17 9 13PP242 10 AURKC (Aurora C) 8 12 10PP242 10 BLK 19 24 22PP242 10 BMX 61 61 61PP242 100 BRAF 19 23 21PP242 100 BRAF V599E 40 37 38PP242 10 BRSK1 (SAD1) 42 42 42PP242 10 BTK 29 31 30PP242 10 CAMK1D (CaMKI delta) 8 7 7PP242 10 CAMK2A (CaMKII alpha) 20 19 20PP242 10 CAMK2B (CaMKII beta) 0 3 2PP242 10 CAMK2D (CaMKII delta) 20 24 22PP242 10 CAMK4 (CaMKIV) 10 14 12PP242 10 CDC42 BPA (MRCKA) -3 0 -1PP242 10 CDC42 BPB (MRCKB) 8 5 6PP242 10 CDK1/cyclin B 9 8 8PP242 10 CDK2/cyclin A 6 9 7PP242 10 CDK5/p35 1 2 2PP242 10 CHEK1 (CHK1) 11 15 13PP242 100 CLK1 14 11 13PP242 10 CLK2 12 7 9PP242 100 CLK3 14 13 13PP242 10 CSF1R (FMS) 4 4 4PP242 10 CSK 16 17 16PP242 10 CSNK1A1 (CK1 alpha 1) -1 -2 -1PP242 10 CSNK1D (CK1 delta) 7 8 7PP242 10 CSNK1E (CK1 epsilon) 46 48 47PP242 10 CSNK1G1 (CK1 gamma 1) 10 11 11PP242 10 CSNK1G2 (CK1 gamma 2) 12 12 12PP242 10 CSNK1G3 (CK1 gamma 3) 13 16 15PP242 10 CSNK2A1 (CK2 alpha 1) 12 13 12PP242 10 CSNK2A2 (CK2 alpha 2) 12 17 14PP242 10 DAPK3 (ZIPK) 25 22 24PP242 100 DCAMKL2 (DCK2) 14 13 14PP242 10 DYRK1A 18 16 17PP242 10 DYRK1B 19 19 19


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP242 10 DYRK3 63 66 64PP242 10 DYRK4 3 4 4PP242 10 EGFR (ErbB1) 7 10 8PP242 10 EGFR L858R (ErbB1 L858R) 24 25 24PP242 10 EGFR L861Q (ErbB1 L861Q) 14 15 14PP242 10 EPHA1 64 61 62PP242 10 EPHA2 42 36 39PP242 10 EPHA3 18 18 18PP242 100 EPHA4 27 26 27PP242 10 EPHA5 37 36 37PP242 10 EPHA8 54 53 53PP242 10 EPHB1 34 33 34PP242 10 EPHB2 36 34 35PP242 10 EPHB3 18 19 19PP242 10 EPHB4 37 38 37PP242 10 ERBB2 (HER2) -2 8 3PP242 10 ERBB4 (HER4) 25 26 25PP242 10 FER 13 15 14PP242 10 FES (FPS) 13 26 19PP242 10 FGFR1 20 27 23PP242 10 FGFR2 21 21 21PP242 10 FGFR3 27 25 26PP242 10 FGFR3 K650E 21 22 22PP242 10 FGFR4 16 21 19PP242 10 FGR 49 53 51PP242 100 FLT1 (VEGFR1) 15 14 15PP242 10 FLT3 35 34 35PP242 10 FLT3 D835Y 64 67 66PP242 10 FLT4 (VEGFR3) 48 48 48PP242 10 FRK (PTK5) 30 34 32PP242 10 FYN 42 41 41PP242 10 GRK4 21 22 22PP242 10 GRK5 3 6 5PP242 10 GRK6 10 8 9PP242 10 GRK7 11 19 15PP242 10 GSK3A (GSK3 alpha) 11 15 13PP242 10 GSK3B (GSK3 beta) 19 17 18PP242 10 HCK 46 45 45PP242 10 HIPK1 (Myak) 22 23 22PP242 10 HIPK4 8 8 8PP242 10 IGF1R 7 5 6PP242 10 IKBKB (IKK beta) 5 1 3PP242 10 INSR 5 2 3PP242 100 INSRR (IRR) 8 9 8PP242 10 IRAK4 4 7 5PP242 10 ITK 13 14 14PP242 10 JAK2 18 20 19PP242 100 JAK2 JH1 JH2 44 42 43PP242 10 JAK2 JH1 JH2 V617F 84 81 82


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP242 10 JAK3 5 8 7PP242 10 KDR (VEGFR2) 59 59 59PP242 100 KIT -3 -2 -2PP242 10 KIT T670I 12 4 8PP242 10 LCK 42 41 41PP242 10 LTK (TYK1) 31 32 32PP242 10 LYN A 43 42 43PP242 10 LYN B 45 40 43PP242 100 MAP2K1 (MEK1) 18 24 21PP242 100 MAP2K2 (MEK2) 18 23 20PP242 100 MAP2K6 (MKK6) 4 15 10PP242 100 MAP3K8 (COT) -23 -10 -17PP242 100 MAP3K9 (MLK1) 22 31 27PP242 10 MAP4K2 (GCK) 67 68 68PP242 10 MAP4K4 (HGK) 52 58 55PP242 10 MAP4K5 (KHS1) 43 38 40PP242 10 MAPK1 (ERK2) 17 19 18PP242 100 MAPK11 (p38 beta) 5 7 6PP242 10 MAPK12 (p38 gamma) 13 13 13PP242 10 MAPK13 (p38 delta) 10 12 11PP242 100 MAPK14 (p38 alpha) 12 15 13PP242 10 MAPK3 (ERK1) 12 8 10PP242 10 MAPKAPK2 -2 1 0PP242 10 MAPKAPK3 7 9 8PP242 10 MAPKAPK5 (PRAK) 7 6 7PP242 10 MARK1 (MARK) 16 14 15PP242 10 MARK2 13 12 12PP242 10 MATK (HYL) -2 -1 -1PP242 10 MERTK (cMER) 10 11 11PP242 10 MET (cMet) 7 7 7PP242 10 MET M1250T 3 4 4PP242 10 MINK1 47 47 47PP242 10 MST1R (RON) 2 1 1PP242 100 MST4 19 8 14PP242 100 MUSK 31 33 32PP242 100 MYLK2 (skMLCK) 5 5 5PP242 10 NEK1 13 18 15PP242 10 NEK2 4 2 3PP242 100 NEK4 13 9 11PP242 10 NEK6 9 9 9PP242 100 NEK7 9 7 8PP242 10 NEK9 5 2 3PP242 100 NTRK1 (TRKA) 5 9 7PP242 10 NTRK2 (TRKB) 8 8 8PP242 10 NTRK3 (TRKC) 39 52 45PP242 10 PAK2 (PAK65) 7 -9 -1PP242 10 PAK3 -10 -1 -5PP242 10 PAK4 4 11 8PP242 10 PAK6 -1 2 0


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP242 10 PAK7 (KIAA1264) 3 2 3PP242 100 PASK 72 77 74PP242 10 PDGFRA (PDGFR alpha) 18 18 18PP242 10 PDGFRA D842V 7 9 8PP242 100 PDGFRA T674I 0 3 2PP242 10 PDGFRB (PDGFR beta) 17 18 17PP242 100 PDK1 9 8 8PP242 10 PHKG1 19 21 20PP242 10 PHKG2 24 27 25PP242 10 PIM1 10 8 9PP242 10 PIM2 2 5 4PP242 10 PKN1 (PRK1) 6 -4 1PP242 10 PLK1 12 13 13PP242 10 PLK2 30 35 32PP242 10 PLK3 35 35 35PP242 10 PRKACA (PKA) 18 19 18PP242 10 PRKCA (PKC alpha) 84 81 82PP242 10 PRKCB1 (PKC beta I) 65 67 66PP242 10 PRKCB2 (PKC beta II) 83 79 81PP242 10 PRKCD (PKC delta) -4 6 1PP242 10 PRKCE (PKC epsilon) 44 44 44PP242 10 PRKCG (PKC gamma) 53 56 55PP242 10 PRKCH (PKC eta) 18 25 22PP242 10 PRKCI (PKC iota) 16 18 17PP242 10 PRKCN (PKD3) 20 17 18PP242 10 PRKCQ (PKC theta) 13 18 15PP242 10 PRKCZ (PKC zeta) 17 17 17PP242 10 PRKD1 (PKC mu) 3 6 5PP242 10 PRKD2 (PKD2) 8 11 9PP242 10 PRKG1 8 5 7PP242 10 PRKG2 (PKG2) 59 57 58PP242 10 PRKX 20 22 21PP242 10 PTK2B (FAK2) 2 2 2PP242 100 PTK6 (Brk) 12 13 12PP242 100 RAF1 (cRAF) 69 69 69PP242 10 RET 89 91 90PP242 10 RET V804L 39 39 39PP242 10 RET Y791F 93 90 92PP242 10 ROCK1 4 -6 -1PP242 10 ROCK2 1 5 3PP242 100 ROS1 18 17 18PP242 10 RPS6KA1 (RSK1) 22 20 21PP242 10 RPS6KA2 (RSK3) 40 42 41PP242 10 RPS6KA3 (RSK2) 33 29 31PP242 10 RPS6KA4 (MSK2) 44 43 44PP242 10 RPS6KA5 (MSK1) 16 12 14PP242 10 RPS6KA6 (RSK4) 51 52 52PP242 10 RPS6KB1 (p70S6K) 14 15 14PP242 10 SGK (SGK1) 12 15 14


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP242 10 SGK2 11 17 14PP242 10 SGKL (SGK3) 19 17 18PP242 10 SRC 32 32 32PP242 10 SRC N1 37 40 38PP242 100 SRMS (Srm) 7 0 3PP242 10 SRPK1 9 8 8PP242 10 SRPK2 4 8 6PP242 10 STK22B (TSSK2) 5 3 4PP242 10 STK22D (TSSK1) 11 15 13PP242 100 STK23 (MSSK1) 10 14 12PP242 100 STK24 (MST3) 18 18 18PP242 10 STK25 (YSK1) 20 26 23PP242 10 STK3 (MST2) 14 18 16PP242 10 STK4 (MST1) 15 17 16PP242 10 STK6 (Aurora A) 32 30 31PP242 10 SYK 23 29 26PP242 100 TAOK2 (TAO1) 7 6 6PP242 10 TBK1 0 1 1PP242 10 TEK (Tie2) 19 20 20PP242 10 TYRO3 (RSE) 25 22 24PP242 10 YES1 65 63 64PP242 10 ZAP70 12 7 9

PP487 10 ABL1 97 95 96PP487 10 ABL1 E255K 99 100 99PP487 10 ABL1 G250E 96 96 96PP487 10 ABL1 T315I 32 34 33PP487 10 ABL1 Y253F 99 97 98PP487 10 ABL2 (Arg) 98 98 98PP487 10 ACVR1B (ALK4) 86 89 88PP487 10 ADRBK1 (GRK2) 4 8 6PP487 10 ADRBK2 (GRK3) 2 2 2PP487 10 AKT1 (PKB alpha) 5 7 6PP487 10 AKT2 (PKB beta) 0 2 1PP487 10 AKT3 (PKB gamma) 16 19 17PP487 10 ALK 26 24 25PP487 10 AMPK A1/B1/G1 (PRKA A1/B1/G1) 40 34 37PP487 10 AURKB (Aurora B) 2 7 5PP487 10 AURKC (Aurora C) 2 3 3PP487 10 BLK 97 97 97PP487 10 BMX 93 95 94PP487 100 BRAF 84 83 83PP487 100 BRAF V599E 93 93 93PP487 10 BRSK1 (SAD1) 40 35 38PP487 10 BTK 96 94 95PP487 10 CAMK1D (CaMKI delta) 18 5 12PP487 10 CAMK2A (CaMKII alpha) -1 2 1PP487 10 CAMK2B (CaMKII beta) 4 6 5PP487 10 CAMK2D (CaMKII delta) 12 20 16


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP487 10 CAMK4 (CaMKIV) 5 10 7PP487 10 CDC42 BPA (MRCKA) 4 5 4PP487 10 CDC42 BPB (MRCKB) 4 1 3PP487 10 CDK1/cyclin B 26 22 24PP487 10 CDK2/cyclin A 8 5 7PP487 10 CDK5/p35 16 10 13PP487 10 CHEK1 (CHK1) 1 1 1PP487 100 CLK1 4 4 4PP487 10 CLK2 56 57 57PP487 100 CLK3 9 8 9PP487 10 CSF1R (FMS) 54 51 53PP487 10 CSK 74 75 75PP487 10 CSNK1A1 (CK1 alpha 1) 39 38 39PP487 10 CSNK1D (CK1 delta) 71 72 72PP487 10 CSNK1E (CK1 epsilon) 74 77 76PP487 10 CSNK1G1 (CK1 gamma 1) -5 -2 -4PP487 10 CSNK1G2 (CK1 gamma 2) 4 0 2PP487 10 CSNK1G3 (CK1 gamma 3) -3 -1 -2PP487 10 CSNK2A1 (CK2 alpha 1) -2 2 0PP487 10 CSNK2A2 (CK2 alpha 2) 10 11 11PP487 10 DAPK3 (ZIPK) 15 14 14PP487 100 DCAMKL2 (DCK2) 3 3 3PP487 10 DYRK1A 13 13 13PP487 10 DYRK1B 11 11 11PP487 10 DYRK3 9 1 5PP487 10 DYRK4 1 0 1PP487 10 EGFR (ErbB1) 70 68 69PP487 10 EGFR L858R (ErbB1 L858R) 84 83 84PP487 10 EGFR L861Q (ErbB1 L861Q) 74 71 72PP487 10 EPHA1 99 97 98PP487 10 EPHA2 88 85 87PP487 10 EPHA3 65 70 68PP487 100 EPHA4 95 95 95PP487 10 EPHA5 91 92 91PP487 10 EPHA8 100 101 101PP487 10 EPHB1 94 94 94PP487 10 EPHB2 96 97 97PP487 10 EPHB3 81 79 80PP487 10 EPHB4 88 87 88PP487 10 ERBB2 (HER2) 38 34 36PP487 10 ERBB4 (HER4) 61 59 60PP487 10 FER 9 10 10PP487 10 FES (FPS) 24 24 24PP487 10 FGFR1 72 75 73PP487 10 FGFR2 69 74 72PP487 10 FGFR3 60 66 63PP487 10 FGFR3 K650E 60 60 60PP487 10 FGFR4 72 73 72PP487 10 FGR 102 101 101


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP487 100 FLT1 (VEGFR1) 84 81 83PP487 10 FLT3 37 40 39PP487 10 FLT3 D835Y 49 49 49PP487 10 FLT4 (VEGFR3) 0 0 0PP487 10 FRK (PTK5) 99 98 99PP487 10 FYN 99 98 99PP487 10 GRK4 -3 -1 -2PP487 10 GRK5 -10 -4 -7PP487 10 GRK6 -6 1 -2PP487 10 GRK7 8 10 9PP487 10 GSK3A (GSK3 alpha) 8 8 8PP487 10 GSK3B (GSK3 beta) 9 9 9PP487 10 HCK 99 99 99PP487 10 HIPK1 (Myak) 2 3 3PP487 10 HIPK4 15 14 15PP487 10 IGF1R 12 15 13PP487 10 IKBKB (IKK beta) 0 2 1PP487 10 INSR 3 0 1PP487 100 INSRR (IRR) 6 6 6PP487 10 IRAK4 2 -2 0PP487 10 ITK 6 6 6PP487 10 JAK2 -1 1 0PP487 100 JAK2 JH1 JH2 -1 -1 -1PP487 10 JAK2 JH1 JH2 V617F 13 12 13PP487 10 JAK3 0 -1 0PP487 10 KDR (VEGFR2) 97 97 97PP487 100 KIT 34 33 34PP487 10 KIT T670I -3 2 0PP487 10 LCK 97 97 97PP487 10 LTK (TYK1) 30 35 32PP487 10 LYN A 97 97 97PP487 10 LYN B 98 98 98PP487 100 MAP2K1 (MEK1) 19 23 21PP487 100 MAP2K2 (MEK2) 35 41 38PP487 100 MAP2K6 (MKK6) 6 4 5PP487 100 MAP3K8 (COT) 10 13 12PP487 100 MAP3K9 (MLK1) 13 16 15PP487 10 MAP4K2 (GCK) 21 25 23PP487 10 MAP4K4 (HGK) 38 39 39PP487 10 MAP4K5 (KHS1) 27 31 29PP487 10 MAPK1 (ERK2) 11 16 13PP487 100 MAPK11 (p38 beta) 57 60 59PP487 10 MAPK12 (p38 gamma) 1 3 2PP487 10 MAPK13 (p38 delta) 7 7 7PP487 100 MAPK14 (p38 alpha) 49 52 50PP487 10 MAPK3 (ERK1) 8 13 10PP487 10 MAPKAPK2 0 -3 -1PP487 10 MAPKAPK3 -2 0 -1PP487 10 MAPKAPK5 (PRAK) 3 4 3


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP487 10 MARK1 (MARK) 9 7 8PP487 10 MARK2 15 6 11PP487 10 MATK (HYL) -5 -5 -5PP487 10 MERTK (cMER) 19 19 19PP487 10 MET (cMet) 5 4 5PP487 10 MET M1250T -2 0 -1PP487 10 MINK1 35 33 34PP487 10 MST1R (RON) -5 -12 -8PP487 100 MST4 6 7 7PP487 100 MUSK 25 24 25PP487 100 MYLK2 (skMLCK) 4 -1 2PP487 10 NEK1 6 -1 2PP487 10 NEK2 9 -5 2PP487 100 NEK4 -1 -2 -1PP487 10 NEK6 4 1 3PP487 100 NEK7 3 1 2PP487 10 NEK9 1 9 5PP487 100 NTRK1 (TRKA) 50 56 53PP487 10 NTRK2 (TRKB) 56 56 56PP487 10 NTRK3 (TRKC) 77 74 75PP487 10 PAK2 (PAK65) 9 6 8PP487 10 PAK3 20 23 22PP487 10 PAK4 8 10 9PP487 10 PAK6 5 6 5PP487 10 PAK7 (KIAA1264) 7 3 5PP487 100 PASK 2 5 3PP487 10 PDGFRA (PDGFR alpha) 89 89 89PP487 10 PDGFRA D842V 108 107 107PP487 100 PDGFRA T674I 10 10 10PP487 10 PDGFRB (PDGFR beta) 82 82 82PP487 100 PDK1 3 3 3PP487 10 PHKG1 4 11 7PP487 10 PHKG2 -7 0 -3PP487 10 PIM1 8 4 6PP487 10 PIM2 9 3 6PP487 10 PKN1 (PRK1) 8 5 6PP487 10 PLK1 8 9 9PP487 10 PLK2 5 6 5PP487 10 PLK3 5 2 3PP487 10 PRKACA (PKA) 12 13 13PP487 10 PRKCA (PKC alpha) 39 25 32PP487 10 PRKCB1 (PKC beta I) 51 45 48PP487 10 PRKCB2 (PKC beta II) 27 31 29PP487 10 PRKCD (PKC delta) 8 11 9PP487 10 PRKCE (PKC epsilon) 17 20 18PP487 10 PRKCG (PKC gamma) 52 54 53PP487 10 PRKCH (PKC eta) 9 13 11PP487 10 PRKCI (PKC iota) 47 62 55PP487 10 PRKCN (PKD3) 31 24 28


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP487 10 PRKCQ (PKC theta) 9 -8 1PP487 10 PRKCZ (PKC zeta) 23 15 19PP487 10 PRKD1 (PKC mu) 19 18 19PP487 10 PRKD2 (PKD2) 28 27 28PP487 10 PRKG1 15 16 15PP487 10 PRKG2 (PKG2) 5 9 7PP487 10 PRKX 19 18 18PP487 10 PTK2B (FAK2) 8 14 11PP487 100 PTK6 (Brk) 100 98 99PP487 100 RAF1 (cRAF) 85 84 84PP487 10 RET 101 101 101PP487 10 RET V804L 28 34 31PP487 10 RET Y791F 100 99 99PP487 10 ROCK1 14 -2 6PP487 10 ROCK2 6 1 3PP487 100 ROS1 53 58 55PP487 10 RPS6KA1 (RSK1) 10 10 10PP487 10 RPS6KA2 (RSK3) 20 23 21PP487 10 RPS6KA3 (RSK2) 7 7 7PP487 10 RPS6KA4 (MSK2) 38 39 39PP487 10 RPS6KA5 (MSK1) 9 12 11PP487 10 RPS6KA6 (RSK4) 24 22 23PP487 10 RPS6KB1 (p70S6K) 14 16 15PP487 10 SGK (SGK1) -2 0 -1PP487 10 SGK2 0 4 2PP487 10 SGKL (SGK3) -2 1 -1PP487 10 SRC 98 99 98PP487 10 SRC N1 99 100 100PP487 100 SRMS (Srm) 94 94 94PP487 10 SRPK1 6 8 7PP487 10 SRPK2 5 8 7PP487 10 STK22B (TSSK2) -4 -2 -3PP487 10 STK22D (TSSK1) 23 19 21PP487 100 STK23 (MSSK1) 2 1 2PP487 100 STK24 (MST3) 26 24 25PP487 10 STK25 (YSK1) 43 45 44PP487 10 STK3 (MST2) 20 23 22PP487 10 STK4 (MST1) 12 9 10PP487 10 STK6 (Aurora A) 22 26 24PP487 10 SYK 2 2 2PP487 100 TAOK2 (TAO1) 7 4 6PP487 10 TBK1 0 5 3PP487 10 TEK (Tie2) 10 11 10PP487 10 TYRO3 (RSE) 88 89 89PP487 10 YES1 99 99 99PP487 10 ZAP70 12 25 19

PP494 10 ABL1 68 69 69PP494 10 ABL1 E255K 57 55 56


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP494 10 ABL1 G250E 46 47 46PP494 10 ABL1 T315I 4 7 6PP494 10 ABL1 Y253F 64 67 65PP494 10 ABL2 (Arg) 48 53 50PP494 10 ACVR1B (ALK4) 51 50 50PP494 10 ADRBK1 (GRK2) 6 3 4PP494 10 ADRBK2 (GRK3) 11 2 6PP494 10 AKT1 (PKB alpha) 2 4 3PP494 10 AKT2 (PKB beta) -1 2 1PP494 10 AKT3 (PKB gamma) 2 6 4PP494 10 ALK 3 8 6PP494 10 AMPK A1/B1/G1 (PRKA A1/B1/G1) 3 3 3PP494 10 AURKB (Aurora B) -2 7 2PP494 10 AURKC (Aurora C) 0 4 2PP494 10 BLK 57 57 57PP494 10 BMX 63 61 62PP494 100 BRAF 43 55 49PP494 100 BRAF V599E 54 59 57PP494 10 BRSK1 (SAD1) 16 15 15PP494 10 BTK 67 66 66PP494 10 CAMK1D (CaMKI delta) -7 17 5PP494 10 CAMK2A (CaMKII alpha) -3 3 0PP494 10 CAMK2B (CaMKII beta) 2 7 4PP494 10 CAMK2D (CaMKII delta) -1 10 4PP494 10 CAMK4 (CaMKIV) -7 2 -2PP494 10 CDC42 BPA (MRCKA) -1 3 1PP494 10 CDC42 BPB (MRCKB) -21 2 -10PP494 10 CDK1/cyclin B 14 22 18PP494 10 CDK2/cyclin A 2 10 6PP494 10 CDK5/p35 12 23 18PP494 10 CHEK1 (CHK1) 0 2 1PP494 100 CLK1 0 -2 -1PP494 10 CLK2 9 12 10PP494 100 CLK3 5 5 5PP494 10 CSF1R (FMS) 51 45 48PP494 10 CSK 26 31 29PP494 10 CSNK1A1 (CK1 alpha 1) 13 10 11PP494 10 CSNK1D (CK1 delta) 19 21 20PP494 10 CSNK1E (CK1 epsilon) 81 88 85PP494 10 CSNK1G1 (CK1 gamma 1) 0 -8 -4PP494 10 CSNK1G2 (CK1 gamma 2) 1 -1 0PP494 10 CSNK1G3 (CK1 gamma 3) -3 -4 -3PP494 10 CSNK2A1 (CK2 alpha 1) -1 4 2PP494 10 CSNK2A2 (CK2 alpha 2) 6 7 6PP494 10 DAPK3 (ZIPK) -1 1 0PP494 100 DCAMKL2 (DCK2) 1 5 3PP494 10 DYRK1A 7 11 9PP494 10 DYRK1B 5 14 10PP494 10 DYRK3 1 -3 -1


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP494 10 DYRK4 1 0 1PP494 10 EGFR (ErbB1) 11 9 10PP494 10 EGFR L858R (ErbB1 L858R) 23 16 19PP494 10 EGFR L861Q (ErbB1 L861Q) 7 8 8PP494 10 EPHA1 92 93 93PP494 10 EPHA2 69 62 65PP494 10 EPHA3 33 35 34PP494 100 EPHA4 61 63 62PP494 10 EPHA5 70 73 72PP494 10 EPHA8 86 85 85PP494 10 EPHB1 66 64 65PP494 10 EPHB2 73 71 72PP494 10 EPHB3 28 25 27PP494 10 EPHB4 64 58 61PP494 10 ERBB2 (HER2) 2 4 3PP494 10 ERBB4 (HER4) 23 18 21PP494 10 FER 1 3 2PP494 10 FES (FPS) 17 21 19PP494 10 FGFR1 15 15 15PP494 10 FGFR2 7 7 7PP494 10 FGFR3 13 6 9PP494 10 FGFR3 K650E 4 1 2PP494 10 FGFR4 10 10 10PP494 10 FGR 86 86 86PP494 100 FLT1 (VEGFR1) 1 -1 0PP494 10 FLT3 9 15 12PP494 10 FLT3 D835Y 11 15 13PP494 10 FLT4 (VEGFR3) -1 -1 -1PP494 10 FRK (PTK5) 67 68 67PP494 10 FYN 85 88 86PP494 10 GRK4 5 -4 1PP494 10 GRK5 2 -11 -4PP494 10 GRK6 4 -4 0PP494 10 GRK7 15 6 10PP494 10 GSK3A (GSK3 alpha) 5 5 5PP494 10 GSK3B (GSK3 beta) 3 3 3PP494 10 HCK 79 79 79PP494 10 HIPK1 (Myak) 1 1 1PP494 10 HIPK4 5 11 8PP494 10 IGF1R 3 -1 1PP494 10 IKBKB (IKK beta) -1 -1 -1PP494 10 INSR 1 -4 -1PP494 100 INSRR (IRR) 1 -3 -1PP494 10 IRAK4 -6 -1 -3PP494 10 ITK 1 -2 -1PP494 10 JAK2 8 -2 3PP494 100 JAK2 JH1 JH2 -5 -2 -4PP494 10 JAK2 JH1 JH2 V617F 2 6 4PP494 10 JAK3 1 -1 0


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP494 10 KDR (VEGFR2) 11 13 12PP494 100 KIT 22 22 22PP494 10 KIT T670I -3 -2 -3PP494 10 LCK 82 85 84PP494 10 LTK (TYK1) 2 5 4PP494 10 LYN A 80 81 80PP494 10 LYN B 80 81 81PP494 100 MAP2K1 (MEK1) 6 6 6PP494 100 MAP2K2 (MEK2) 5 6 5PP494 100 MAP2K6 (MKK6) 1 2 1PP494 100 MAP3K8 (COT) 9 12 11PP494 100 MAP3K9 (MLK1) -1 1 0PP494 10 MAP4K2 (GCK) -3 5 1PP494 10 MAP4K4 (HGK) 10 19 15PP494 10 MAP4K5 (KHS1) 8 15 11PP494 10 MAPK1 (ERK2) 4 11 8PP494 100 MAPK11 (p38 beta) 9 12 10PP494 10 MAPK12 (p38 gamma) 3 4 3PP494 10 MAPK13 (p38 delta) 3 6 4PP494 100 MAPK14 (p38 alpha) -9 1 -4PP494 10 MAPK3 (ERK1) 3 5 4PP494 10 MAPKAPK2 -1 -2 -2PP494 10 MAPKAPK3 -2 0 -1PP494 10 MAPKAPK5 (PRAK) 3 4 3PP494 10 MARK1 (MARK) 1 1 1PP494 10 MARK2 3 4 4PP494 10 MATK (HYL) -5 -6 -5PP494 10 MERTK (cMER) 4 6 5PP494 10 MET (cMet) -6 -1 -4PP494 10 MET M1250T -4 -2 -3PP494 10 MINK1 15 18 17PP494 10 MST1R (RON) -13 -13 -13PP494 100 MST4 -6 5 0PP494 100 MUSK 0 -2 -1PP494 100 MYLK2 (skMLCK) -3 4 1PP494 10 NEK1 27 35 31PP494 10 NEK2 -2 8 3PP494 100 NEK4 0 4 2PP494 10 NEK6 5 2 3PP494 100 NEK7 0 2 1PP494 10 NEK9 -4 9 3PP494 100 NTRK1 (TRKA) 53 47 50PP494 10 NTRK2 (TRKB) 49 46 47PP494 10 NTRK3 (TRKC) 70 64 67PP494 10 PAK2 (PAK65) 1 7 4PP494 10 PAK3 0 -11 -6PP494 10 PAK4 -8 2 -3PP494 10 PAK6 0 4 2PP494 10 PAK7 (KIAA1264) 0 7 4


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP494 100 PASK -3 6 2PP494 10 PDGFRA (PDGFR alpha) 15 13 14PP494 10 PDGFRA D842V 10 11 10PP494 100 PDGFRA T674I -5 10 3PP494 10 PDGFRB (PDGFR beta) 17 10 14PP494 100 PDK1 1 3 2PP494 10 PHKG1 0 8 4PP494 10 PHKG2 -3 -3 -3PP494 10 PIM1 1 9 5PP494 10 PIM2 -4 7 2PP494 10 PKN1 (PRK1) 0 11 6PP494 10 PLK1 0 -1 0PP494 10 PLK2 -1 8 3PP494 10 PLK3 12 5 8PP494 10 PRKACA (PKA) 1 2 1PP494 10 PRKCA (PKC alpha) -3 14 5PP494 10 PRKCB1 (PKC beta I) 12 13 12PP494 10 PRKCB2 (PKC beta II) 3 13 8PP494 10 PRKCD (PKC delta) -6 5 -1PP494 10 PRKCE (PKC epsilon) 4 14 9PP494 10 PRKCG (PKC gamma) 9 16 13PP494 10 PRKCH (PKC eta) -13 2 -6PP494 10 PRKCI (PKC iota) -6 -2 -4PP494 10 PRKCN (PKD3) 11 12 11PP494 10 PRKCQ (PKC theta) 3 4 4PP494 10 PRKCZ (PKC zeta) 5 15 10PP494 10 PRKD1 (PKC mu) 13 17 15PP494 10 PRKD2 (PKD2) 11 16 13PP494 10 PRKG1 8 11 9PP494 10 PRKG2 (PKG2) 6 10 8PP494 10 PRKX 12 11 11PP494 10 PTK2B (FAK2) 0 -1 -1PP494 100 PTK6 (Brk) 95 96 96PP494 100 RAF1 (cRAF) 74 71 72PP494 10 RET 59 62 61PP494 10 RET V804L 6 4 5PP494 10 RET Y791F 60 62 61PP494 10 ROCK1 -6 7 1PP494 10 ROCK2 -3 4 1PP494 100 ROS1 5 7 6PP494 10 RPS6KA1 (RSK1) 1 2 1PP494 10 RPS6KA2 (RSK3) 11 14 12PP494 10 RPS6KA3 (RSK2) 3 4 4PP494 10 RPS6KA4 (MSK2) 4 4 4PP494 10 RPS6KA5 (MSK1) 9 8 8PP494 10 RPS6KA6 (RSK4) 6 11 8PP494 10 RPS6KB1 (p70S6K) 12 17 14PP494 10 SGK (SGK1) -1 1 0PP494 10 SGK2 0 5 2


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AveragePP494 10 SGKL (SGK3) -3 1 -1PP494 10 SRC 66 66 66PP494 10 SRC N1 70 68 69PP494 100 SRMS (Srm) 17 13 15PP494 10 SRPK1 2 11 7PP494 10 SRPK2 3 10 6PP494 10 STK22B (TSSK2) 1 1 1PP494 10 STK22D (TSSK1) 3 4 3PP494 100 STK23 (MSSK1) 1 1 1PP494 100 STK24 (MST3) 0 13 7PP494 10 STK25 (YSK1) -14 4 -5PP494 10 STK3 (MST2) 0 13 6PP494 10 STK4 (MST1) -10 5 -3PP494 10 STK6 (Aurora A) 3 0 2PP494 10 SYK 5 1 3PP494 100 TAOK2 (TAO1) -5 3 -1PP494 10 TBK1 1 5 3PP494 10 TEK (Tie2) 0 1 0PP494 10 TYRO3 (RSE) 9 6 8PP494 10 YES1 84 86 85PP494 10 ZAP70 14 10 12

S1 10 ABL1 99 98 99S1 10 ABL1 E255K 98 98 98S1 10 ABL1 G250E 96 96 96S1 10 ABL1 T315I 12 15 14S1 10 ABL1 Y253F 97 98 98S1 10 ABL2 (Arg) 97 98 97S1 10 ACVR1B (ALK4) 76 75 75S1 10 ADRBK1 (GRK2) 6 8 7S1 10 ADRBK2 (GRK3) 2 3 2S1 10 AKT1 (PKB alpha) 10 16 13S1 10 AKT2 (PKB beta) 9 17 13S1 10 AKT3 (PKB gamma) 15 21 18S1 10 ALK 63 65 64S1 10 AURKB (Aurora B) 9 10 10S1 10 BLK 93 94 94S1 10 BMX 82 85 84S1 100 BRAF 55 52 53S1 100 BRAF V599E 75 83 79S1 10 BTK 87 89 88S1 10 CAMK1D (CaMKI delta) 5 7 6S1 10 CAMK2A (CaMKII alpha) 1 8 5S1 10 CAMK2B (CaMKII beta) 17 16 16S1 10 CAMK2D (CaMKII delta) 31 40 36S1 10 CAMK4 (CaMKIV) -2 2 0S1 10 CDK1/cyclin B 10 20 15S1 10 CDK2/cyclin A 28 30 29S1 10 CDK5/p35 4 8 6


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS1 10 CHEK1 (CHK1) 12 15 13S1 10 CHEK2 (CHK2) -7 7 0S1 100 CLK1 2 2 2S1 10 CLK2 21 25 23S1 10 CSF1R (FMS) 64 70 67S1 10 CSK 68 68 68S1 10 CSNK1A1 (CK1 alpha 1) 49 51 50S1 10 CSNK1D (CK1 delta) 57 59 58S1 10 CSNK1E (CK1 epsilon) 71 73 72S1 10 CSNK1G1 (CK1 gamma 1) 3 7 5S1 10 CSNK1G2 (CK1 gamma 2) 9 19 14S1 10 CSNK1G3 (CK1 gamma 3) 13 17 15S1 10 CSNK2A1 (CK2 alpha 1) -1 5 2S1 10 CSNK2A2 (CK2 alpha 2) 13 5 9S1 10 DAPK3 (ZIPK) -1 -3 -2S1 10 DYRK3 1 2 2S1 10 DYRK4 -3 0 -1S1 10 EGFR (ErbB1) 28 29 28S1 10 EGFR L858R (ErbB1 L858R) 46 46 46S1 10 EGFR L861Q (ErbB1 L861Q) 35 31 33S1 10 EPHA1 99 99 99S1 10 EPHA2 86 90 88S1 10 EPHA3 80 80 80S1 100 EPHA4 90 91 91S1 10 EPHA5 97 96 97S1 10 EPHA8 85 84 85S1 10 EPHB1 92 94 93S1 10 EPHB2 92 93 92S1 10 EPHB3 90 92 91S1 10 EPHB4 94 92 93S1 10 ERBB2 (HER2) 17 17 17S1 10 ERBB4 (HER4) 8 13 11S1 10 FER 26 33 30S1 10 FES (FPS) 32 38 35S1 10 FGFR1 70 71 71S1 10 FGFR2 76 76 76S1 10 FGFR3 38 41 40S1 10 FGFR4 59 61 60S1 10 FGR 98 99 99S1 100 FLT1 (VEGFR1) 68 68 68S1 10 FLT3 76 84 80S1 10 FLT3 D835Y 90 95 92S1 10 FLT4 (VEGFR3) 78 80 79S1 10 FRK (PTK5) 100 98 99S1 10 FYN 100 101 100S1 10 GRK4 5 3 4S1 10 GRK5 -1 -5 -3S1 10 GRK6 5 7 6S1 10 GRK7 5 9 7


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS1 10 GSK3A (GSK3 alpha) 7 11 9S1 10 GSK3B (GSK3 beta) 9 11 10S1 10 HCK 100 99 99S1 10 IGF1R 57 62 60S1 10 IKBKB (IKK beta) 8 10 9S1 10 INSR 12 18 15S1 100 INSRR (IRR) 8 8 8S1 10 IRAK4 32 44 38S1 10 ITK 7 14 10S1 10 JAK2 16 14 15S1 10 JAK3 6 10 8S1 10 KDR (VEGFR2) 95 97 96S1 100 KIT 32 35 33S1 10 KIT T670I 7 15 11S1 10 LCK 91 93 92S1 10 LYN A 96 96 96S1 10 LYN B 95 96 95S1 100 MAP2K1 (MEK1) 11 22 17S1 100 MAP2K2 (MEK2) 56 58 57S1 100 MAP2K6 (MKK6) 0 2 1S1 100 MAP3K8 (COT) 51 64 57S1 100 MAP3K9 (MLK1) 7 10 8S1 10 MAP4K2 (GCK) 52 56 54S1 10 MAP4K4 (HGK) 38 46 42S1 10 MAP4K5 (KHS1) 39 45 42S1 10 MAPK1 (ERK2) -1 6 2S1 100 MAPK11 (p38 beta) 18 18 18S1 10 MAPK12 (p38 gamma) 10 15 12S1 10 MAPK13 (p38 delta) 12 17 15S1 100 MAPK14 (p38 alpha) 39 40 39S1 10 MAPK3 (ERK1) 14 20 17S1 10 MAPKAPK2 2 0 1S1 10 MAPKAPK3 9 10 10S1 10 MAPKAPK5 (PRAK) 7 8 7S1 10 MATK (HYL) 3 9 6S1 10 MERTK (cMER) 8 12 10S1 10 MET (cMet) 9 28 19S1 10 MET M1250T 1 5 3S1 10 MINK1 36 33 34S1 10 MST1R (RON) -6 4 -1S1 100 MST4 54 53 53S1 100 MUSK 2 5 3S1 100 MYLK2 (skMLCK) -2 -5 -4S1 10 NEK1 31 45 38S1 10 NEK2 11 18 15S1 100 NTRK1 (TRKA) 68 69 68S1 10 NTRK2 (TRKB) 71 72 72S1 10 NTRK3 (TRKC) 78 82 80S1 10 PAK3 11 29 20


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS1 10 PAK4 11 20 15S1 10 PAK6 13 16 14S1 100 PASK 9 13 11S1 10 PDGFRA (PDGFR alpha) 68 72 70S1 10 PDGFRA D842V 82 85 84S1 100 PDGFRA T674I 5 13 9S1 10 PDGFRB (PDGFR beta) 67 69 68S1 100 PDK1 7 11 9S1 10 PHKG1 13 19 16S1 10 PHKG2 20 20 20S1 10 PIM1 15 20 18S1 10 PIM2 9 8 9S1 10 PKN1 (PRK1) -1 7 3S1 10 PLK1 4 5 4S1 10 PLK2 3 11 7S1 10 PLK3 0 4 2S1 10 PRKACA (PKA) 13 39 26S1 10 PRKCA (PKC alpha) 41 44 42S1 10 PRKCB1 (PKC beta I) 19 27 23S1 10 PRKCB2 (PKC beta II) 20 22 21S1 10 PRKCD (PKC delta) 38 44 41S1 10 PRKCE (PKC epsilon) 47 51 49S1 10 PRKCG (PKC gamma) 66 67 66S1 10 PRKCH (PKC eta) 77 80 78S1 10 PRKCI (PKC iota) 16 19 17S1 10 PRKCN (PKD3) 33 31 32S1 10 PRKCQ (PKC theta) 31 36 33S1 10 PRKCZ (PKC zeta) 15 19 17S1 10 PRKD1 (PKC mu) 25 25 25S1 10 PRKD2 (PKD2) 27 34 31S1 10 PRKG1 16 22 19S1 10 PRKG2 (PKG2) 24 30 27S1 10 PRKX 13 14 14S1 10 PTK2 (FAK) 94 94 94S1 10 PTK6 (Brk) 99 96 97S1 100 RAF1 (cRAF) 47 48 47S1 10 RET 101 101 101S1 10 ROCK1 33 35 34S1 10 ROCK2 67 75 71S1 100 ROS1 11 11 11S1 10 RPS6KA1 (RSK1) 4 4 4S1 10 RPS6KA2 (RSK3) 4 2 3S1 10 RPS6KA3 (RSK2) -3 0 -2S1 10 RPS6KA4 (MSK2) 15 23 19S1 10 RPS6KA5 (MSK1) 8 14 11S1 10 RPS6KB1 (p70S6K) 11 9 10S1 10 SGK (SGK1) -1 9 4S1 10 SGK2 4 16 10S1 10 SGKL (SGK3) 4 10 7


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS1 10 SRC 98 98 98S1 10 SRC N1 100 101 101S1 10 SRC N2 96 96 96S1 100 SRMS (Srm) 39 38 38S1 10 SRPK2 -1 1 0S1 10 STK22B (TSSK2) 10 12 11S1 10 STK22D (TSSK1) 16 18 17S1 100 STK24 (MST3) 52 54 53S1 10 STK25 (YSK1) 84 84 84S1 10 STK3 (MST2) 64 71 68S1 10 STK4 (MST1) 71 69 70S1 10 STK6 (Aurora A) 17 16 16S1 10 SYK 13 20 17S1 100 TAOK2 (TAO1) 5 8 6S1 10 TBK1 1 4 2S1 10 TEK (Tie2) 9 12 11S1 10 TYRO3 (RSE) 36 42 39S1 10 YES1 99 99 99S1 10 ZAP70 10 7 9

S2 10 ABL1 77 79 78S2 10 ABL1 E255K 58 61 60S2 10 ABL1 G250E 45 50 47S2 10 ABL1 T315I 29 33 31S2 10 ABL1 Y253F 77 81 79S2 10 ABL2 (Arg) 54 61 58S2 10 ACVR1B (ALK4) 51 54 52S2 10 ADRBK1 (GRK2) -1 3 1S2 10 ADRBK2 (GRK3) -1 -1 -1S2 10 AKT1 (PKB alpha) 6 13 10S2 10 AKT2 (PKB beta) -2 10 4S2 10 AKT3 (PKB gamma) 3 21 12S2 10 ALK 5 18 11S2 10 AURKB (Aurora B) 15 22 18S2 10 BLK 56 63 60S2 10 BMX 65 72 68S2 100 BRAF 48 58 53S2 100 BRAF V599E 83 80 82S2 10 BTK 41 55 48S2 10 CAMK1D (CaMKI delta) 11 9 10S2 10 CAMK2A (CaMKII alpha) -4 2 -1S2 10 CAMK2B (CaMKII beta) 20 10 15S2 10 CAMK2D (CaMKII delta) 13 21 17S2 10 CAMK4 (CaMKIV) 6 0 3S2 10 CDK1/cyclin B 0 2 1S2 10 CDK2/cyclin A 1 -5 -2S2 10 CDK5/p35 0 -1 0S2 10 CHEK1 (CHK1) 34 27 30S2 10 CHEK2 (CHK2) 0 13 7


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS2 100 CLK1 -4 -1 -3S2 10 CLK2 -1 6 2S2 10 CSF1R (FMS) 38 50 44S2 10 CSK 31 37 34S2 10 CSNK1A1 (CK1 alpha 1) 3 6 5S2 10 CSNK1D (CK1 delta) 61 67 64S2 10 CSNK1E (CK1 epsilon) 97 98 97S2 10 CSNK1G1 (CK1 gamma 1) -1 2 1S2 10 CSNK1G2 (CK1 gamma 2) 0 9 4S2 10 CSNK1G3 (CK1 gamma 3) 3 9 6S2 10 CSNK2A1 (CK2 alpha 1) -2 4 1S2 10 CSNK2A2 (CK2 alpha 2) 0 1 0S2 10 DAPK3 (ZIPK) -2 -2 -2S2 10 DYRK3 0 0 0S2 10 DYRK4 -1 -2 -1S2 10 EGFR (ErbB1) 55 58 56S2 10 EGFR L858R (ErbB1 L858R) 73 74 74S2 10 EGFR L861Q (ErbB1 L861Q) 65 70 68S2 10 EPHA1 77 82 79S2 10 EPHA2 55 66 61S2 10 EPHA3 26 23 25S2 100 EPHA4 62 63 63S2 10 EPHA5 75 80 77S2 10 EPHA8 82 85 83S2 10 EPHB1 75 81 78S2 10 EPHB2 73 82 77S2 10 EPHB3 40 51 46S2 10 EPHB4 70 71 70S2 10 ERBB2 (HER2) 14 15 15S2 10 ERBB4 (HER4) -2 16 7S2 10 FER 4 12 8S2 10 FES (FPS) 22 17 20S2 10 FGFR1 58 60 59S2 10 FGFR2 44 49 47S2 10 FGFR3 24 27 25S2 10 FGFR4 30 35 33S2 10 FGR 78 84 81S2 100 FLT1 (VEGFR1) 11 15 13S2 10 FLT3 11 23 17S2 10 FLT3 D835Y 2 10 6S2 10 FLT4 (VEGFR3) 34 38 36S2 10 FRK (PTK5) 54 66 60S2 10 FYN 71 78 75S2 10 GRK4 2 5 3S2 10 GRK5 -1 -6 -3S2 10 GRK6 4 5 5S2 10 GRK7 4 6 5S2 10 GSK3A (GSK3 alpha) 2 3 2S2 10 GSK3B (GSK3 beta) -3 5 1


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS2 10 HCK 75 82 79S2 10 IGF1R -3 10 3S2 10 IKBKB (IKK beta) 5 10 8S2 10 INSR -9 4 -3S2 100 INSRR (IRR) -6 -2 -4S2 10 IRAK4 -1 19 9S2 10 ITK -4 1 -2S2 10 JAK2 8 1 5S2 10 JAK3 5 0 2S2 10 KDR (VEGFR2) 80 83 82S2 100 KIT 29 30 30S2 10 KIT T670I -5 5 0S2 10 LCK 78 85 82S2 10 LYN A 72 84 78S2 10 LYN B 81 85 83S2 100 MAP2K1 (MEK1) 6 10 8S2 100 MAP2K2 (MEK2) 38 46 42S2 100 MAP2K6 (MKK6) -5 -11 -8S2 100 MAP3K8 (COT) -11 4 -4S2 100 MAP3K9 (MLK1) 2 11 6S2 10 MAP4K2 (GCK) 39 45 42S2 10 MAP4K4 (HGK) 84 87 86S2 10 MAP4K5 (KHS1) 45 50 47S2 10 MAPK1 (ERK2) -14 3 -5S2 100 MAPK11 (p38 beta) 5 9 7S2 10 MAPK12 (p38 gamma) -1 9 4S2 10 MAPK13 (p38 delta) 2 10 6S2 100 MAPK14 (p38 alpha) 31 44 37S2 10 MAPK3 (ERK1) 3 13 8S2 10 MAPKAPK2 -8 1 -4S2 10 MAPKAPK3 1 7 4S2 10 MAPKAPK5 (PRAK) 2 6 4S2 10 MATK (HYL) 11 18 14S2 10 MERTK (cMER) 13 22 17S2 10 MET (cMet) 4 7 6S2 10 MET M1250T -5 -1 -3S2 10 MINK1 73 81 77S2 10 MST1R (RON) -10 -1 -5S2 100 MST4 5 16 10S2 100 MUSK -15 -1 -8S2 100 MYLK2 (skMLCK) 8 12 10S2 10 NEK1 43 57 50S2 10 NEK2 9 21 15S2 100 NTRK1 (TRKA) -4 3 -1S2 10 NTRK2 (TRKB) 1 6 3S2 10 NTRK3 (TRKC) -4 8 2S2 10 PAK3 -3 1 -1S2 10 PAK4 14 15 14S2 10 PAK6 13 12 13


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS2 100 PASK 6 10 8S2 10 PDGFRA (PDGFR alpha) 55 60 57S2 10 PDGFRA D842V 19 26 23S2 100 PDGFRA T674I -1 7 3S2 10 PDGFRB (PDGFR beta) 21 24 23S2 100 PDK1 -5 2 -2S2 10 PHKG1 2 11 7S2 10 PHKG2 14 18 16S2 10 PIM1 14 16 15S2 10 PIM2 10 9 10S2 10 PKN1 (PRK1) -11 -8 -9S2 10 PLK1 -4 1 -1S2 10 PLK2 2 8 5S2 10 PLK3 1 3 2S2 10 PRKACA (PKA) 28 33 31S2 10 PRKCA (PKC alpha) 10 16 13S2 10 PRKCB1 (PKC beta I) 10 21 15S2 10 PRKCB2 (PKC beta II) 10 20 15S2 10 PRKCD (PKC delta) -1 -2 -1S2 10 PRKCE (PKC epsilon) 11 10 10S2 10 PRKCG (PKC gamma) 24 30 27S2 10 PRKCH (PKC eta) 1 9 5S2 10 PRKCI (PKC iota) 6 8 7S2 10 PRKCN (PKD3) 68 66 67S2 10 PRKCQ (PKC theta) 9 14 11S2 10 PRKCZ (PKC zeta) 3 4 3S2 10 PRKD1 (PKC mu) 65 57 61S2 10 PRKD2 (PKD2) 76 66 71S2 10 PRKG1 4 6 5S2 10 PRKG2 (PKG2) 13 13 13S2 10 PRKX 8 10 9S2 10 PTK2 (FAK) 40 49 45S2 10 PTK6 (Brk) 86 87 86S2 100 RAF1 (cRAF) 63 66 64S2 10 RET 79 82 81S2 10 ROCK1 4 7 6S2 10 ROCK2 9 9 9S2 100 ROS1 -3 -2 -3S2 10 RPS6KA1 (RSK1) 0 1 0S2 10 RPS6KA2 (RSK3) 0 1 1S2 10 RPS6KA3 (RSK2) -2 6 2S2 10 RPS6KA4 (MSK2) 1 17 9S2 10 RPS6KA5 (MSK1) 0 9 4S2 10 RPS6KB1 (p70S6K) 8 7 7S2 10 SGK (SGK1) -1 11 5S2 10 SGK2 2 13 8S2 10 SGKL (SGK3) 0 10 5S2 10 SRC 46 53 50S2 10 SRC N1 67 69 68


Drug [ATP] (μM) Kinase Assay 1 Assay 2 AverageS2 10 SRC N2 54 61 57S2 100 SRMS (Srm) 4 9 6S2 10 SRPK2 2 0 1S2 10 STK22B (TSSK2) -3 2 0S2 10 STK22D (TSSK1) -10 -3 -6S2 100 STK24 (MST3) 6 14 10S2 10 STK25 (YSK1) 2 6 4S2 10 STK3 (MST2) -1 1 0S2 10 STK4 (MST1) 5 8 6S2 10 STK6 (Aurora A) 14 16 15S2 10 SYK -2 8 3S2 100 TAOK2 (TAO1) 0 1 0S2 10 TBK1 3 11 7S2 10 TEK (Tie2) 7 15 11S2 10 TYRO3 (RSE) 31 36 34S2 10 YES1 78 85 82S2 10 ZAP70 6 2 4

Supplementary Table 2. SelectScreen profiling data for selected pyrazolopyrimidine inhibitors. Complete data for reference compounds is available at http://www.invitrogen.com/downloads/SelectScreen_Data_193.pdf.

Supplementary Table 3 a. Data collection and refinement statistics for c-Src complex structures

PDB code Compound

3EN4 PP121

3EN5 PP494

3EN6 PP102

3EN7 S1

Data collection Space group P1 P1 P1 P1 Cell dimensions a, b, c (Å) 42.37, 63.14, 73.94 42.30, 62.99, 74.14 42.44, 63.05, 74.58 42.43, 62.98, 74.03 α, β, γ (°) 100.63, 88.68, 90.40 101.14, 92.15, 90.12 101.31, 91.17, 89.62 100.84, 91.81, 89.61 Resolution (Å) 50.00–2.55 (2.64-

2.55) * 50.00–2.65 (2.74–2.65) *

50.00–2.40 (2.49–2.40) *

50.00–2.80 (2.90–2.80) *

Rmerge 10.1 (47.2) 15.3 (41.7) 8.2 (36.6) 12.4 (49.7) I / σI 12.4 (2.4) 9.6 (2.4) 14.5 (2.6) 11.8 (2.8) Completeness (%) 98.4 (95.0) 98.4 (97.0) 96.1 (81.1) 98.8 (98.2) Redundancy 3.8 (3.3) 3.6 (2.6) 3.7 (2.9) 3.8 (3.6) Refinement Resolution (Å) 42.36–2.55 43.15–2.66 43.22–2.39 37.13–2.81 No. reflections 22721 20103 27021 17102 Rwork / Rfree 19.8 / 25.5 20.4 / 24.8 21.0 / 25.9 20.7 / 25.7 No. atoms Protein 4048 3820 3711 3774 Ligand/ion 48 48 23 20 Water 6 14 15 6 B-factors Protein 25.76 13.98 27.93 14.82 Ligand/ion 43.24 25.24 50.59 29.84 Water 18.73 9.01 24.29 4.85 R.m.s. deviations Bond lengths (Å) 0.011 0.014 0.016 0.013 Bond angles (°) 1.339 1.601 1.659 1.538

Data for each structure collected from a single crystal. *Highest-resolution shell is shown in parentheses. Ramachandran outliers for c-Src complex structures were calculated with RAMPAGE1. Ramachandran statistics for each complex are as follows:

PDB code Compound

3EN4 PP121

3EN5 PP494

3EN6 PP102

3EN7 S1

Number of residues in favored region 499 (97.3%) 467 (95.9%) 447 (97.8%) 449 (96.8%) Number of residues in allowed region 14 (2.7%) 19 (3.9%) 10 (2.2%) 13 (2.8%) Number of residues in outlier region 0 (0.0%) 1 (0.2%) 0 (0.0%) 2 (0.4%)

1 S. C. Lovell, I. W. Davis, W. B. Arendall et al., Proteins 50 (3), 437 (2003).

b. Data collection and refinement statistics for p110γ structures Compound Data collection statistics

S2 S1

Wavelengtha

Resolution

0.97930 2.4 Å

0.97930 2.5 Å

Completeness (last shell) Rmerge b (last shell) Multiplicity (last shell)

99.3 (99.3) 0.07 (0.36) 3.7 (3.7)

99.7 (97.4) 0.05 (0.32) 3.7 (3.8)

<I/σ> (last shell) 6.8 (2.0) 10.0 (1.7) Refinement statistics

Resolution

57.1-2.4 Å

54.4-2.5 Å

Number of reflections, (no cutoff) 38512 33672 Protein atoms 6846 6850 Waters 38 49 Rcryst c

Rfree c (% data used) r.m.s.d. from idealityd bonds/angles/dihedrals Average B (Wilson B factor)

0.23 0.27 (4.1) 0.016/1.501/7.023 25 (47)

0.25 0.30 (4.1) 0.014/1.457/7.157 21 (59)

RMSD B for bonded main (side) chain atoms 0.5 (1.6) 0.5 (1.4) aData sets were collected at ESRF beamline ID14-4

bRmerge = ∑hkl∑i |Ii(hkl) - <I(hkl)>| / ∑hkl∑i Ii(hkl).

cRcryst and Rfree = ∑ |Fobs - Fcalc| / ∑ Fobs; Rfree calculated with the percentage of the data

shown in parenthesis

dr.m.s. deviations for bond angles and lengths in regard to Engh and Huber parameters

Supplementary Table 3. Crystallographic statistics for Src and p110γ structures. See Supplementary Experimental Methods for additional details.

Supplementary Movie: Conformational changes in Src associated with PP121 binding. This movie shows four successive crystal structures of Src, illustrating the conformational movements associated with binding of the drugs PP102, PP494, and PP121, and in the final part, activation of the kinase. In the first structure (PP102 bound), helix C is disordered and not visible at the right of the screen. In the second structure (PP494 bound), helix C is in an inactive conformation. Binding of PP121 in the third structure results in movement of helix C into an active conformation in order to satisfy the hydrogen bond between the PP121 azaindole and the side chain of Glu310. The final structure shows the hydrogen bond between the catalytic lysine (Lys295) o and Glu310 and the conformation of helix C when Src is in an active conformation.

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Supplementary Experimental Methods 1. Kinase Assays 1.1 p110α, p110β, p110γ, p110δ: Inhibitors were tested against recombinant PI3-Ks (Upstate or Jena Biosciences) at two-fold dilutions (concentration range: 50 μM - 0.003 μM) in an assay containing 25 mM HEPES, pH 7.5, 10 mM MgCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Freshly sonicated phosphatidylinositol (Sigma; 1 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.2 PI4-KIIIβ: Inhibitors were tested against recombinant PI4-KIIIβ at two-fold dilutions (concentration range: 50 μM - 0.003 μM) in an assay containing 50 mM Tris, pH 7.5, 20 mM MgCl2, 0.4% Triton X-100, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Freshly sonicated phosphatidylinositol (Sigma; 1 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.3 DNA-PK: Inhibitors were tested against DNA-PK (Promega) at two-fold dilutions (concentration range: 50 μM - 0.003 μM) in an assay containing 50 mM HEPES, pH 7.5, 1mM EGTA, 10 mM MgCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Dephosphorylated casein (Sigma; 10 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.4 mTOR (FRAP1): Inhibitors were tested against recombinant mTOR (Invitrogen) at two-fold dilutions (concentration range: 50 μM - 0.003 μM) in an assay containing 50 mM HEPES, pH 7.5, 1mM EGTA, 10 mM MgCl2, 2.5 mM, 0.01% Tween, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Rat recombinant PHAS-1/4EBP1 (Calbiochem; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.5 c-Src, Src(T338I), and Hck: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant kinase in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. The peptide substrate EIYGEFKKK was used as phosphoacceptor (200 μM) . Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging.

2

IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.6 c-Abl: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant c-Abl in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. The peptide substrate EAIYAAPFAKKK was used as phosphoacceptor (200 μM) . Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.7 VEGFR2 (KDR): Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant KDR receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.8 EphB4 receptor: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant Ephrin receptor B4 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.9 EGF receptor: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant EGF receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.1% BME, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.10 c-KIT: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant KIT kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 1mM DTT, 10mM MnCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were

3

calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.11 RET: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant RET kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2.5mM DTT, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. The peptide substrate EAIYAAPFAKKK was used as phosphoacceptor (200 μM) . Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.12 PDGFRβ: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant PDGF receptor β kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2.5mM DTT,10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. The peptide substrate EAIYAAPFAKKK was used as phosphoacceptor (200 μM) . Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.13 FLT-3: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant FLT-3 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2.5mM DTT,10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. The optimized Abl peptide substrate EAIYAAPFAKKK was used as phosphoacceptor (200 μM) . Reactions were terminated by spotting onto phosphocellulose sheets, which were washed with 0.5% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 1.14 TIE2: Inhibitors were tested at two-fold dilutions (concentration range: 50 μM - 0.003 μM) against recombinant TIE2 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2mM DTT, 10mM MnCl2, 10 μM ATP (2.5 μCi of γ-32P-ATP), and 3 μg/mL BSA. Poly E-Y (Sigma; 2 mg/mL) was used as a substrate. Reactions were terminated by spotting onto nitrocellulose, which was washed with 1M NaCl/1% phosphoric acid (approximately 6 times, 5-10 min each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging. IC50 values were calculated by fitting the data to a sigmoidal dose-response curve using the Prism software package. 2. SelectScreen profiling of kinase inhibitors Compounds were assayed by Invitrogen at a final drug concentration of 1 μM and, typically, an ATP concentration of 10 μM. See Supplementary Table 2 for complete data, and http://www.invitrogen.com/downloads/PAC-SSBK.pdf for complete assay details. Complete data for reference kinase inhibitors is available at http://www.invitrogen.com/downloads/SelectScreen_Data_193.pdf.

4

3. Principle component analysis Selectivity data was available for 172 pyrazolopyrimidines and eight reference compounds against 13 kinases (p110α, p110β, p110γ, p110δ, DNA-PK, mTOR, Src, SrcT338I, Abl, Hck, VEGFR2, EGFR, EphB4). A 180x13 table was generated containing the log IC50 values for each of these compounds against these targets and this table was input into Matlab's principle component analysis function. The first two principle components, representing 51.9% and 21.7% of the total variation in the data, are plotted in Figure 1c. This graph provides a two-dimensional representation of the 13-dimensional relationship between the selectivity profiles of these compounds. Compounds closer in this space will tend to have more similar selectivity profiles. 4. Determination of p110γ crystal structures 5.1 Protein expression and purification: Recombinant human p110γ (residues 144-1102, with a His6 tag directly fused to the C-terminus) was purified from baculovirus-infected Sf9 cells. Cells were sonicated in buffer A (0.1M NaCl, 0.005M potassium phosphate pH 8 (4° C), 10 mM Tris-Hcl pH 8 4°C) and 1 mM MgCl2) and the supernatant from a 1 hr ultracentrifugation was purified by heparin affinity, metal chelate affinity and gel filtration. The protein in gel filtration buffer (20 mM Tris pH 7.2 (4°C), 0.5 mM ammonium sulfate, 1% ethylene glycol, 0.02% CHAPS and 5 mM DTT) was concentrated to approximately 6 mg/ml. 4.2 Crystallisation of p110γ and soaking with inhibitors: Crystals were grown at 17 ºC using sitting-drop vapour-diffusion by mixing 1 μl p110γ sample (4 mg/mL in a buffer containing 0.5 mM (NH4)2SO4, 20 mM Tris pH 7.2, 1% ethylene glycol, 0.02% Chaps and 5 mM DTT) and 1 μL of a reservoir solution (16-17% PEG 4000, 250 mM (NH4)2SO4 and 100mM Tris pH 7.5). Crystal seeds were introduced in the drops by hair seeding with a cat whisker. The crystals grow over 1-2 weeks reaching a maximum size of 0.2 mm x 0.1 mm x 0.1 mm.

Inhibitor stocks were diluted in freezing solution (23% PEG 4000, 250 mM (NH4)2SO4, 100mM Tris pH 7.5 and 14% glycerol) to final concentrations of 0.01 mM, 0.1 mM and 1 mM. Aliquots of increasing inhibitor concentration in freezing solution were added to the drops in which the crystals were grown. The additions were of 0.5 μL and drops were incubated for 30-60 min between additions. Finally, 1 μL was taken out of the drop and 1 μL of 1 mM inhibitor in freezing solution was added, and the crystals were soaked in this solution for three hours. After soaking, the crystals were transferred to a fresh drop containing 1mM inhibitor in freezing solution then immediately frozen by dunking the crystals in liquid nitrogen. 4.3 Diffraction data collection and structure refinement: Diffraction data were collected at ESRF ID14-4. Data were integrated with MOSFLM and scaled using SCALA. Human p110γ was used as an initial model for molecular replacement using AMORE. The model was refined using REFMAC, starting with rigid-body refinement then using restrained maximum-likelihood refinement with individual isotropic B factors alternated with restrained refinement using TLS parameters. Cycles of REFMAC refinement were alternated with manual rebuilding. The data collection and refinement statistics are summarised in Supplementary Table 4.

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5. Determination of c-Src crystal structures 5.1 Protein expression, purification, and crystallisation: The chicken c-Src kinase domain (residues 251–533) was expressed, purified and crystallized as described.1 M. A. Seeliger and J. Kuriyan provided the chicken c-Src gene cloned into pSKB-32 and a plasmid containing tyrosine phosphatase YopH3. G. Montelione provided a plasmid containing GroEL/Trigger Factor (cloned into pACYADuet-1, Novagen). Inhibitor stock solutions were prepared by dissolving each inhibitor in DMSO to 5 mM (S1) or 20 mM (PP102, PP121 and PP494). Each inhibitor was added in a 1.5 molar excess to aliquots of concentrated c-Src protein solutions (14–26 mg mL–1 c-Src, 50 mM Tris (pH 8.0), 100 mM NaCl, 5% (v/v) glycerol, 1 mM DTT). After incubation at room temperature for 30 min, these mixtures were centrifuged for 10 min at 15,000 × g, and the clear supernatants were used for crystallization. Cocrystals were grown using hanging-drop vapor diffusion with a 1:1 ratio mixture of protein and 18–22% (w/v) ethylene glycol precipitant solutions. Crystals of space group P1 grew within one week as thin plates3. For each cocrystal structure, the following conditions were used:

c-Src complex [Protein] (mg mL–1)

[Precipitant] % ethylene glycol (w/v)

Hanging drop volume (µL)

PP121 14 20 2 PP102 14 20 2

S1 16 18 3 PP494 26 22 2

The c-Src cocrystals were cryo-protected using 20% glycerol before flash-freezing the crystals in liquid nitrogen. 5.2 Diffraction data collection and structure refinement: The diffraction data were collected at the Advanced Light Source (Berkeley, California, United States) beamlines 8.2.1 and 8.2.2 under a nitrogen gas stream at 100 K, using a wavelength of 1.0000 nm. The data were processed with DENZO and SCALEPACK4 using the HKL2000 program suite. Chicken c-Src cocrystal structures were solved by molecular replacement with Phaser5 starting coordinates where taken from the catalytic domain of human c-Src (PDB entry 1yoj3). Topology and parameter files for the inhibitors were generated using the Dundee PRODRG26 Server (http://davapc1.bioch.dundee.ac.uk/programs/prodrg/). Refinement of the structures was carried out through multiple cycles of manual fitting using Coot and crystallographic refinement using refmac58. Crystallographic refinement involved TLS refinement followed by restrained refinement with the maximum likelihood method. During the initial rounds of manual fitting and refinement, optimal TLS groups were determined with the TLSMD9 web server (http://skuld.bmsc.washington.edu/~tlsmd/). For the later rounds of fitting and refinement, two TLS groups were used – one for the N-lobe (residues 248–342) and one for the C-lobe (residues 343–533). Ramachandran outliers were calculated with RAMPAGE10. Detailed data and refinement statistics are available in Supplementary Table 4. Ramachandran statistics for each complex are as follows: c-Src complex PP121 PP102 S1 PP494 Number of residues in favored region

506 (97.9%) 446 (97.2%) 452 (97.4%) 477 (97.9%)

Number of residues in allowed 11 (2.1%) 13 (2.8%) 11 (2.4%) 10 (2.1%)

6

region Number of residues in outlier region 0 (0.0%) 0 (0.0%) 1 (0.1%) 0 (0.0%)

6. Alignment and analysis of PI3K-γ and c-Src structures: The kinase structures were structurally aligned in UCSF Chimera11 using Scheeff and Bourne’s manual structural alignment12 of 31 kinases (http://www.sdsc.edu/pb/kinases/). Since the structure of VEGFR-2 (1vr2)13 was not included in Scheeff and Bourne’s alignment, we created our own alignment of the “universal” core of 1vr2 to 2src (c-Src) and 1e8x (p110γ) using the reported method.12 To order allow the best direct comparison between the protein kinases and p110γ, 1vr2 and the structures reported here were aligned against 1e8x (p110γ) using the residues for the “universal” core as defined by Scheeff and Bourne.12 Likewise, the root mean square deviation (RMSD) of the aligned Cα backbone of 1vr2, the c-Src/BA121 complex and 1e8x was calculated using the residues for the “universal” core except the activation loop residues in all three structures and the kinase insert domain residues of 1vr2. These residues are often disordered or missing in kinase crystal structures, thereby contributing to an unusually high RMSD. The crystallographic figures were produced using PyMOL.14 To compare the relative binding orientations of pyrazolopyrimidine containing inhibitors in p110γ versus c-Src, the p110γ and c-Src inhibitor complexes were structurally aligned based on the adenine ring of ATP. The adenine ring atoms of ATP in 1e8x and ANP in 2src were aligned using the PyMOL pair-fitting tool. Using the UCSF Chimera11 MatchMaker tool, the p110γ/S2 and p110γ/S1 complexes were aligned against the adenine-aligned 1e8x structure. Similarly, we aligned the c-Src/PP121, c-Src/PP102, c-Src/S1 and c-Src/PP494 complexes against the adenine-aligned 2src structure. 7. Tumour cell western blotting: U87 and LN229 cells were grown in 12-well plates in DMEM containing 10% FBS and penicillin/streptomycin. Cells were treated for 1 h with either inhibitor at four-fold dilutions between 10 μM and 0.040 μM or vehicle (0.1% DMSO), then lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were resolved by SDS-PAGE, transferred to nitrocellulose by electroblotting, and probed for the indicated proteins. All antibodies were from Cell Signaling Technology. 8 Tumour cell proliferation assays 8.1 Tumour cell proliferation assays - IC50 values: All tumour cell lines were grown in media as recommended by the ATCC. Cells were plated in 96 well plates to approximately 30% confluence in serum with 0.5%, 2% or 10% FBS and left overnight to adhere. The following day cells were treated with inhibitor at four-fold dilutions in appropriate serum concentration so that the final inhibitor concentration was between 10 μM and 0.040 μM or vehicle (0.1% DMSO). After 72 hours of treatment 10μl of 440μM Resazurin sodium salt (Sigma) was added to each well. When a difference in color became detectable by eye (2-24 hours) florescence intensity in each well was measured using a top-reading florescent plate reader. 8.2 Tumour cell proliferation assays - single cell counting: Cells were treated with drug (2.5 μM) or vehicle (0.1% confluence). Once each day, cells were diluted into trypan blue and viable cells were counted using a

7

hemocytometer. Each experiment was performed in quadruplicate. Cells were diluted prior to reaching confluence and drug was replaced every 48 h. 9. Cell cycle analysis by flow cytometry: Cells were grown to 60% confluence in 6-well plates containing 10% fetal bovine serum (FBS). Media was removed, cells were washed with PBS and treated with drug (2.5 μM) or vehicle (0.1% DMSO) in media plus 10% FBS. After 24 h of incubation at 37ºC, cells were trypsinized, harvested and fixed with cold EtOH (70%). Cells were treated with RNase and stained with propidium iodide, then sorted using a FACS Calibur flow cytometer (Becton Dickinson). Results were analyzed and cell cycle stage of cells in samples were determined using Modifit-LT software (Verity Software). 10. Endothelial cell western blotting: HUVECs were grown in 12-well plates in complete media (Clonetics). Cells were then serum starved for 8 h in basal media (Clonetics); pretreated with either inhibitor at four-fold dilutions between 10 μM and 0.040 μM or vehicle (0.1% DMSO) for 30 min; and then stimulated with VEGF (4 ng/mL) for 30 min Cells were then lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were resolved by SDS-PAGE, transferred to nitrocellulose by electroblotting, and probed for the indicated proteins. All antibodies were from Cell Signaling Technology. 11. Endothelial Cell Tube Formation Assays: In vitro angiogenesis was assayed using the Endothelial Cell Tube Formation Assay (CellBiolabs) as described by the manufacturer’s protocol. Briefly, a 96 well plate was coated with ECM gel (50 μL/well). ECM gel was solidified at 37º C and HUVECs (Clonetics) were plated on the matrix (1.4 x 104 cells/well) in complete media (Clonetics) with appropriate concentration of PP121, PP102, PI-103, PIK-90 or sorafenib. After 24 h, cells were treated with Calcein AM and four images/well were obtained using a fluorescent microscope. Tube length was quantified using ImageJ software. 12. Apoptosis analysis by flow cytometry Suspension cells (BaF3 and K562 cell lines) were split to 20% confluence in 10cm plates containing media with 10% fetal bovine serum (FBS). Cells were then treated with drug (2.5 μM or 5 μM) or vehicle (0.1% DMSO). After 36-72 hours of incubation at 37ºC, cells were harvested, strained, washed and resuspended in AnnexinV buffer (100 μL; 10mM HEPES, 140mM NaCl and 2.5 mM CaCl2, pH 7.4). 5 μL of FITC-AnnexinV (Invitrogen) and 1 μL propidium iodide (250 μg/mL in PBS) was added to the cells and the cells were left in the dark to incubate for 20 minutes. of AnnexinV buffer (400 μL) was added to each sample and the florescence of cell populations was determined with a FACS Calibur flow cytometer (Becton Dickinson) using CellQuest Pro software. Results were analyzed using FlowJo 7.2.4 Software. 13. Morphological reversion of Src expressing cells. NIH-3T3 cells expressing v-Src (T338) were grown on fibronectin coated coverslips to 50% confluence. Cells were treated with DMSO, PP1, PP121 or PI-103 (2.5 μM) for 24 hours. Cells were fixed for 20 minutes at room temperature with 4% formaldehyde in cytoskeleton buffer with sucrose (CBS: 10 mM MES (pH 6.1), 138 mM KCl, 3 mM MCl2, 2 mM EGTA and 320 mM sucrose). Cells were then washed with TBS, permeabilized in TBS-0.5% Triton-X-100 (TBS-TX) for 10 minutes, then blocked with TBS-TX with 2% BSA for 10 minutes. Coverslips were incubated for 20 min with Alexa Fluor 546 phalloidin (1 μg/mL) and DAPI (5 μg/mL) for 20 minutes. Cells were then

8

washed, coverslips were mounted on slides and imaged on a Zeiss Axiovert 200M florescence microscope using AxioVision Rel. 4.6 software. For analysis, 4 images per coverslip were taken with a Zeiss EC Plan-NEO FLUAR 40X/0.75 objective. For each condition, approximately 100 cells were scored for the presence of actin stress fibers. The scorer was blinded to the identity of each sample. Images shown in figure were taken with a Zeiss Plan-Apochromat 63X/1.4 Oil Dic objective. 14. Isolation of kinase inhibitors from commercial tablets 12.1 Gefitinib: Four 250 mg Iressa tablets (AstraZeneca) were crushed and added to water to create a suspension. 1N HCl was added until the suspension reached pH 2. The remaining insoluble material was removed by Buchner filtration to give a clear filtrate, which was basified to pH 10 using saturated aqueous Na2CO3. The product was extracted EtOAc (5x), organic phases were combined, dried with Na2SO4, and concentrated in vacuo to yield 942 mg (94 %) gefitinib free base. The proton NMR data of the recovered solid matched the patent literature values for the monohydrate form of erlotinib: 1H NMR (DMSO-d6) δ 9.51 (s, 1H), 8.45 (s, 1H), 8.07 (dd, J=6.8, 2.6 Hz, 1H), 7.76–7.72 (m, 2H), 7.40 (t, J=9.2 Hz, 1H), 7.16 (s, 1H), 4.14 (t, J=6.4 Hz, 2H), 3.89 (s, 3H), 3.53 (t, J=4.6 Hz, 4H), 2.45–2.32 (m, obscured by water peak, 6H), 1.95 (m, 2H); MS (ESI) m/z 447.1 [M + H]+. 12.2 Dasatinib: Ten 70 mg tablets of Sprycel (Bristol-Myers Squibb) were crushed and mixed with DMF (20 mL). The suspension was sonicated for 30 min at room temperature, filtered through Celite, and concentrated in vacuo to yield a white powder. 25 mL of 14.5:10.5:2.5 MeOH:EtOAc:2 M aq. NH4OH was added to the solid and the resulting suspension was sonicated at room temperature for 30 min, filtered through celite and concentrated in vacuo to yield a white solid. Crude product was purified by the following recrystallization: crude product was dissolved in 5 mL 80 % EtOH/dH2O, with heating. The solution was cooled, then dH2O was added dropwise until the solution turned cloudy. The solution was left at room temperature until crystal formation was complete (30 min). The solid was filtered and rinsed with dH2O to yield a white powder at approximately 40% yield. The proton NMR data of the recovered crystals matched the patent literature values for the monohydrate form of dasatinib: 1H NMR (DMSO-d6) δ 11.45 (s, 1H), 9.86 (s, 1H), 8.21 (s, 1H), 7.40 (dd, J=7.3, 1.5 Hz, 1H), 7.29 (m, 1H), 7.25 (app. t, 1H), 6.05 (s, 1H), 4.43 (t, J=5.3 Hz, 1H), 3.56–3.47 (m, 6H), 2.47 (m, 4H), 2.45–2.38 (m, 5H), 2.24 (s, 3H).

Acknowledgements We thank A. Dar (Shokat lab, UCSF) for help with protein crystallography, M. A. Seeliger and J. Kuriyan (UC Berkeley) and G. Montelione (Rutgers) for reagents, L. Kelly and M.S. Madhusudhan (Sali lab, UCSF) for discussions on structure-based alignments and the staff and funding agencies of beamlines 8.2.1 and 8.2.2 (Advanced Light Source, Berkeley, CA) for their assistance with x-ray diffraction data collection. NIH P41 RR-01081 supports UCSF Chimera software development. 1 J. A. Blair, D. Rauh, C. Kung et al., Nat. Chem. Biol. 3 (4), 229 (2007). 2 M. A. Seeliger, M. Young, M. N. Henderson et al., Protein Sci. 14 (12), 3135 (2005). 3 C. B. Breitenlechner, N. A. Kairies, K. Honold et al., J. Mol. Biol. 353 (2), 222 (2005). 4 Z. Otwinowski and W. Minor, Meth. Enzymol. 276A, 307 (1997).

9

5 A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams et al., J. Appl. Crystallogr. 40 (4), 658 (2007).

6 A. W. Schuttelkopf and D. M. van Aalten, Acta Crystallogr. 60 (Pt 8), 1355 (2004). 7 P. Emsley and K. Cowtan, Acta Crystallogr. D Biol. Crystallogr. 60 (Pt 12 Pt 1), 2126

(2004). 8 G. N. Murshudov, A. A. Vagin, and E. J. Dodson, Acta Crystallogr. D Biol. Crystallogr. 53

(Pt 3), 240 (1997). 9 J. Painter and E. A. Merritt, J. Appl. Crystallogr. 39 (1), 109 (2006). 10 S. C. Lovell, I. W. Davis, W. B. Arendall et al., Proteins 50 (3), 437 (2003). 11 E. F. Pettersen, T. D. Goddard, C. C. Huang et al., J. Comput. Chem. 25 (13), 1605

(2004). 12 E. D. Scheeff and P. E. Bourne, PLoS Comput. Biol. 1 (5), e49 (2005). 13 M. A. McTigue, J. A. Wickersham, C. Pinko et al., Structure 7 (3), 319 (1999). 14 W. L. DeLano, The PyMOL Molecular Graphics Program (DeLano Scientific, Palo Alto,

CA, USA, 2002).

10

Supplementary Synthetic methods All RP-HPLC was carried out on either a Varian ProStar solvent delivery system or a Ranin SD-200 solvent delivery system each equipped with a Zorbax 300-SB C18 column. The column was eluted with CH3CN/H2O/0.1%TFA (1-100% gradient), which was monitored by UV at λ=260 nm. 1H and 13C NMR were recorded on a Varian Innova 400 spectrometer at 400 MHz and 100 MHz, respectively. 1H chemical shifts are reported in δ (ppm) as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broad) and are referenced to the residual solvent peak. Low resolution electrospray ionization LC/MS (ESI-MS) were recorded on a Waters Micromass ZQ equipped with a Waters 2695 Separations module using an XTerra MS C18 3.6μm column (Waters).

Synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA18)

A solution of 250 mL of formamide and 3-amino-4-pyrazolecarbonitrile (25 g, 0.231 mol) was heated to 180°C overnight under an argon atmosphere. Reaction was cooled and 400 mL of dH2O were added. The resulting solid was filtered and rinsed with cold dH2O. White solid precipitate was collected and dried in vacuo overnight to yield BA18 (39 g, 100% yield). 1H NMR (DMSO-d6) δ 8.13 (s, 1H), 8.07 (s, 1H), 7.6 (br); 13C NMR (DMSO-d6) δ 163.5, 158.6, 156.4, 133.1, 100.2; ESI-MS (M+H)+ m/z calcd 136.1, found 136.1. Synthesis of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA19)

A solution of 3H-pyrazolo[3,4-d]pyrimidin-4-amine (10g, 0.074 mol) and n-iodo-succinamide (25 g, 0.111 mol) in DMF (80mL) was heated to 80°C overnight under an argon atmosphere. The resulting solid was filtered and rinsed with cold EtOH. Product was dried in vacuo overnight to yield BA19 (24 g, 100% yield). 1H NMR (DMSO-d6) δ 11.06 (br, 1H), 8.17 (s, 1H); 13C NMR (DMSO-d6) δ 157.6, 156.0, 155.2, 102.5, 89.8; ESI-MS (M+H)+ m/z calcd 262.0, found 262.0 Synthesis of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP12)

A solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2 g, 0.0077 mol) and K2CO3 (4.2g, 0.031 mol) in DMF (50 mL) was brought to 80°C under an argon atomosphere. Isopropylbromide (1.0g, 0.0084 mol) was added with a syringe. Reaction was refluxed under argon atmosphere for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Sodium citrate (50 mL) was added and reaction was extracted with EtOAc. Organic phases concentrated in vacuo and purified using silica gel column chromatography [MeOH—CH2Cl2, 5:95] yielding PP12 (1.68 g, 72% yield). 1H NMR (CDCl3) δ 8.31 (s, 1H), 5.99 (br, 2H), 5.07 (m, J=6.6 Hz,

N

N NNH

NH2

NNH

NC

H2N N

N NNH

NH2 I

N

N NN

NH2 I

18 19 12

11

1H); 13C NMR (CDCl3) δ 157.5, 155.9, 153.2, 104.2, 85.6, 49.9, 22.2; ESI-MS (M+H)+ m/z calcd 304.0, found 304.1. ESI-HRMS (M+H)+ m/z calcd 304.0059, found 304.0049.

Synthesis of 4-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-benzenesulfonamide (PP14)

A solution of benzenesulfonamide-4-boronic acid pinacol ester (23 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP14 (2.2 mg, 10% yield). ESI-MS (M+H)+ m/z calcd 333.1, found 333.1. ESI-HRMS (M+H)+ m/z calcd 333.1134, found 333.1126.

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP15)

A solution of 2 methoxy-4-(4,4,5,5-tetramethyl-1,3-2-dioxaborolan-2-yl) phenol (19 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP15 (4.3 mg, 20% yield). 1H NMR (DMSO-d6) δ 9.39 (br), 8.34 (s, 1H), 7.16 (d, J=2.3 Hz, 1H), 7.08 (dd, J=8.0 Hz, J=2.1 Hz, 1H), 6.939 (d, J=8.0 Hz, 1H); ESI-MS (M+H)+ m/z calcd 300.1, found 300.2. ESI-HRMS (M+H)+ m/z calcd 300.1461, found 300.1462.

N

N NN

NH2 I

12

N

N NN

NH2

SO

ONH2

14

N

N NN

NH2 I

12 15

N

N NN

NH2

OH

OMe

12

Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)naphthalen-2-ol (PP17)

A solution of 6-hydroxynaphthalen-2-yl-2-boronic acid (15 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP17 (4.8 mg, 23% yield). 1H NMR (CD3OD) δ 8.37 (s, 1H), 8.08 (s, 1H), 7.86 (d, J=8.6 Hz, 2H), 7.71 (dd, J=8.7 Hz, J=1.5 Hz, 1H), 7.22 (d, J=2.4Hz, 1H), 7.18 (dd, J=8.9, J=2.4, 1H), 5.24 (m, J=6.6 Hz, 1H), 1.62 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 320.1, found 320.1. ESI-HRMS (M+H)+ m/z calcd 320.1511, found 320.1499.

Synthesis of tert-butyl 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylcarbamate (PP20)

A solution of 4-N-Boc-amino-3-methoxy-benzeneboronic acid (48 mg, 0.18 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.18 mmol) in DME (12 mL). Pd(PPh3)4 (40 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP20. 1H NMR (CDCl3) δ 8.22 (s, 1H), 7.82 (d, J=8.3 Hz, 1H), 7.64 (s, 1H), 7.32 (s, 1H), 6.37 (br), 5.29 (m, J=6.7 Hz, 1H),

N

N NN

NH2 I

12

N

N NN

NH2

17

OH

N

N NN

NH2 I

12

N

N NN

NH2

HN

OMe

20

O

O

13

4.00 (s, 3H), 1.63 (d, J=6.6 Hz, 6H), 1.53 (s, 9H); ESI-MS (M+H)+ m/z calcd 399.2, found 399.1.

Synthesis of 3-(4-amino-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP202)

A solution of tert-butyl 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylcarbamate (PP20) (20 mg, 0.05 mmol) in CH2Cl2, TFA, S(CH2)2, H2O (45:45:5:5) (1mL) was stirred at room temperature for 15 minutes. NaHCO3 (2 mL) was added till reaction was alkaline. Reaction was extrated with H2O and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP202. ESI-HRMS (M+H)+ m/z calcd 299.1620, found 299.1609.

Synthesis of 2-amino-5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (PP203)

PP20 (tert-butyl 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylcarbamate, 7 mg, 0.018 mmol) was dissolved in CH2Cl2 (2.5 mL) and stirred under an argon atmosphere at room temperature. BBr3 (0.500 mL) was added slowly with a syringe. The reaction mixture was stirred overnight, under argon at room temperature. BBr3 was removed in vacuo and the remaining solid was purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP203.

N

N NN

NH2

NH2

OMe

202

N

N NN

NH2

HN

OMe

20

O

O

N

N NN

NH2

NH2

OH

203

N

N NN

NH2

HN

OMe

20

O

O

14

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)pyridine-2-carbonitrile (PP21)

A solution of 2-cyanopyridine 5-boronic acid pinocol ester (18 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP21 (2.5 mg, 14% yield). 1H NMR (CDCl3) δ 9.06 (d, J=1.43 Hz, 1H), 8.30 (s, 1H), 8.18 (dd, J=1.73 Hz, J=1.73 Hz, 1H), 7.92 (d, J=8.5 Hz, 1H), 5.26 (m, J=6.50 Hz, 1H), 1.64 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 280.1, found 280.1. ESI-HRMS (M+H)+ m/z calcd 280.1311, found 280.1299.

Synthesis of 3-(3-(benzyloxy)-5-fluorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA22).

A solution of (3-Benzyloxy-5-fluorophenyl)boronic acid (29 mg, 5.80 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield 15.6 mg (60% yield). ESI-MS (M+H)+ m/z calcd 378.1, found 378.0. Synthesis of 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-fluorophenol (PP22)

N

N NN

NH2 I

12

N

N NN

NH2

N

21

N

N

N NN

NH2 I

12

N

N NN

NH2

N

N NN

NH2

22

FOBn

FOH

15

A solution of3 -(3-(benzyloxy)-5-fluorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA22, 15 mg, 0.04 mmol) in MeOH (0.9 mL) was flushed with argon. Pd on activated carbon (10 mL) was carefully added while keeping reaction under an argon atmosphere. Reaction was flushed with H2 gas and left under H2 atmosphere overnight at room temperature. The reaction was filtered through celite and rinsed with MeOH to yield PP22 (15 mg, 100% yield). 1H NMR (CD3OD) δ 8.37 (s, 1H), 6.96 (s, 1H), 6.92 (d, J=9.0 Hz, 1H), 6.70 (d, J=10.5 Hz, 1H), 5.21 (m, J=6.7 Hz, 1H), 1.61 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 288.1, found 288.1; ESI-HRMS (M+H)+ m/z calcd 288.1275, found 288.1275.

Synthesis of 1-isopropyl-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP23)

A solution of 3,4-Dimethoxyphenylboronic acid (24 mg, 0.13 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP23 (13.1 mg, 60% yield). 1H NMR (CDCl3) δ 8.24 (s, 1H), 7.15 (m, 2H), 7.04 (m, 1H), 5.19 (m, J=7.1Hz, 1H), 3.97 (s, 6H), 1.64 (d, J=6.6 Hz, 6H) ; ESI-MS (M+H)+ m/z calcd 314.0, found 314.1; ESI-HRMS (M+H)+ m/z calcd 314.1617, found 314.1632.

Synthesis of tert-butyl 2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-(benzyloxy)-1H-indole-1-carboxylate (BA24)

A solution of 5-Benzyloxy-1-BOC-indole-2-boronic acid (303mg, 0.83 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-

N

N NN

NH2 I

12

N

N NN

NH2

OMe

OMe

23

N

N NN

NH2 I

12

N

N NN

NH2 N

N

N NN

NH2 NH

BnO

O

O

HO

BA24 242

16

d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [EtOAc—hexanes, 5:95] to yield BA24. ESI-MS (M+H)+ m/z calcd 499.2, found 499.2. Synthesis of 2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol (PP242)

PP242 (3-(4-fluoro-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 30 mg, 0.10 mmol) was dissolved in a solution of formic acid (4.5 mL, 10 equivalents) and HCl (0.45 mL, 1 equivalent). The reaction was heated and stirred for one hour under an argon atmosphere. The reaction was then concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP242. ESI-MS (M+H)+ m/z calcd 309.1, found 309.1.

Synthesis of (3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)methanol (PP26)

A solution of (3-Hydroxymethylphenyl)boronic acid (24 mg, 0.13 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP26 (8.4 mg, 42% yield). 1H NMR (CDCl3) δ 11.53 (br), 8.22 (s, 1H), 7.71 (s, 1H), 7.56-7.53 (m, 3H), 6.29 (br), 5.20 (m, J=6.6 Hz, 1H), 4.81 (s, 2H), 1.63 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 283.1, found 284.2, ESI-HRMS (M+H)+ m/z calcd 284.1511, found 284.1519.

N

N NN

NH2 I

12

N

N NN

NH2

26

OH

N

N NN

NH2 I

12

N

N NN

NH2

30

NH

O

S

N

17

Synthesis of 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(4,5-dihydrothiazol-2-yl)benzamide (PP30)

A solution of [3-((4,5-dihydrothiazol-2-yl)carbamoyl)phenyl]boronic acid (19 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP30 (17.8 mg, 67% yield). ESI-HRMS (M+H)+ m/z calcd 382.1450, found 382.1467.

Synthesis of 1-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)ethanone (PP31)

A solution of 4-Acetylphenylboronic acid (12.7 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP31 (12.9 mg, 62% yield). 1H NMR (CD3OD) δ 8.33 (s, 1H), 8.21 (dt, J=8.6 Hz, 2.1 Hz, 2H), 7.89 (dt, J=8.0 Hz, J=1.4 Hz, 2H), 5.21 (m, J=6.9 Hz, 1H), 2.71 (s, 3H), 1.62 (d, J=7.0 Hz, 6H).

Synthesis of N-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenyl)acetamide (PP32)

N

N NN

NH2 I

12

N

N NN

NH2

31

O

N

N NN

NH2 I

12

N

N NN

NH2

Cl

32

NH

O

18

A solution of (4-Aminocarbonyl-3-chlorophenyl)boronic acid (16 mg, 0.08 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP32 (9.7 mg, 42% yield). 1H NMR (CD3OD) δ 8.41 (s, 1H), 7.85 (m, 1H), 7.23 (m, 1H), 5.26 (m, J=6.6 Hz, 1H), 1.63 (d, J=7.1 Hz, 6H); ESI-MS (M+H)+ m/z calcd 331.1, found 331.1, ESI-HRMS (M+H)+ m/z calcd 331.1074, found 331.1085.

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-3-methylthiophene-2-carbaldehyde (PP34)

A solution of 5-Formyl-3-methylthiophene-2-boronic acid (26 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP34 (14.7 mg, 38% yield). 1H NMR (CDCl3) δ 9.48 (s, 1H), 7.80 (s, 1H), 6.80 (s, 1H), 4.77 (m, J=6.7 Hz, 1H), 1.92 (s, 3H), 1.17 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 302.1, found 302.0; ESI-HRMS (M+H)+ m/z calcd 302.1076, found 302.1076.

N

N NN

NH2 I

12

N

N NN

NH2

34

S

CHOMe

N

N NN

NH2 I

12

N

N NN

NH2

35

O

OHC

19

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)furan-3-carbaldehyde (PP35)

A solution of 4-Formylfuran-2-boronic acid (20 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP35 (13.5 mg, 39% yield). 1H NMR (CDCl3) δ 8.24 (s, 1H), 7.59 (s, 1H), 7.08 (s, 1H), 5.49 (s, 1H), 5.09 (m, J=6.6 Hz, 1H), 1.54 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 272.1, found 272.1; ESI-HRMS (M+H)+ m/z calcd 272.1147, found 272.1157.

Synthesis of N-[3-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-phenyl]-methanesulfonamide (PP38)

A solution of 3-Methanesulfonylaminophenylboronic acid (32 mg, 0.15 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (16 mg, 0.014 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP38 (24.3 mg, 54% yield). 1H NMR (CD3OD) δ 8.36 (s, 1H), 7.63 (m, 1H), 7.56 (m, 1H), 7.38 (m, 1H), 5.23 (m, J=6.4 Hz, 1H), 3.05 (s, 3H), 1.60 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 347.1, found 347.0; ESI-HRMS (M+H)+ m/z calcd 347.1290, found 347.1302.

Synthesis of 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzonitrile (PP39)

A solution of 3-Cyanophenylboronic acid (23 mg, 0.15 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3

N

N NN

NH2 I

12

N

N NN

NH2

HN

38

SO

O Me

N

N NN

NH2 I

12

N

N NN

NH2

39

N

20

(1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP39 (14.9 mg, 41% yield). 1H NMR (CDCl3) δ 8.26 (s, 1H), 8.02 (s, 1H), 7.86 (m, J=9.1, 2H), 7.7 (t, J=7.9, 1H), 5.22 (m, J=6.6 Hz, 1H), 1.63 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 279.1, found 279.0; ESI-HRMS (M+H)+ m/z calcd 279.1358, found 279.1367.

Synthesis of N-[4-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-phenyl]-methanesulfonamide (PP40)

A solution of 4-Methanesulfonylaminophenylboronic acid (24 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP40 (0.9 mg, 3% yield). ESI-MS (M+H)+ m/z calcd 347.1, found 347.0; ESI-HRMS (M+H)+ m/z calcd 347.1290, found 347.1290.

Synthesis of 3-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-benzenesulfonamide (PP41)

A solution of Benzenesulfonamide-3-boronic acid pinacol ester (31 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with

N

N NN

NH2 I

12

N

N NN

NH2

40

NHS

O

OMe

N

N NN

NH2 I

12

N

N NN

NH2

41

S NH2

O

O

21

saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP41 (9.2 mg, 28% yield). ESI-MS (M+H)+ m/z calcd 333.1, found 333.0; ESI-HRMS (M+H)+ m/z calcd 333.1134, found 333.1134.

Synthesis of 2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)benzo[b]thiophene-5-carbaldehyde (PP42)

A solution of 5-Formylbenzo[b]thiophene-2-boronic acid pinacol ester (31 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP42 (15.2 mg, 45% yield). 1H NMR (CD3OD) δ 8.38 (s, 1H), 8.02 (s, 1H), 7.96 (d, J=8.6 Hz, 1H), 7.78 (s, 1H), 7.51 (dd, J=8.6 Hz, J=1.5 Hz, 1H), 5.53 (s, 1H), 5.23 (m, J=7.1 Hz, 1H), 1.61 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 338.1, found 338.0; ESI-HRMS (M+H)+ m/z calcd 338.1076, found 338.1084.

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-3-carbaldehyde (PP43)

A solution of N-Boc-3-formyl-5-indoleboronic acid pinacol ester (40 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and

N

N NN

NH2 I

12

N

N NN

NH2

42

S

OHC

N

N NN

NH2 I

12

N

N NN

NH2

43

HN

CHO

22

purified by RP-HPLC (MeCN:H2O:0.1% TFA). The TFA from purification hydrolyzed the Boc to yield PP43. 1H NMR (CDCl3) δ 10.11 (s, 1H), 9.03 (br), 8.66 (s, 1H), 8.23 (s, 1H), 7.99 (d, J=2.8 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.3 Hz, J=1.7 Hz, 1H), 5.22 (m, J=6.7 Hz, 1H), 1.65 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 321.1, found 321.0; ESI-HRMS (M+H)+ m/z calcd 321.1464, found 321.1475.

Synthesis of 3-(benzo[c][1,2,5]oxadiazol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP44)

A solution of Benzo[c][1,2,5]oxadiazole-5-boronic acid (18 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP44. 1H NMR (CDCl3) δ 8.32 (s, 1H), 8.14 (t, J=1.7 Hz, 1H), 8.08 (dd, J=9.0 Hz, J=0.7 Hz, 1H), 7.86 (dd, J=9.4 Hz, J=1.2 Hz, 1H), 5.26 (m, J=6.6 Hz, 1H), 1.64 (d, J=7.03 Hz, 6H); ESI-MS (M+H)+ m/z calcd 296.1, found 296.1; ESI-HRMS (M+H)+ m/z calcd 296.1260, found 296.1260.

Synthesis of 2-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)acetonitrile (PP45)

A solution of (4-Cyanomethylphenyl)boronic acid (18 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an

N

N NN

NH2 I

12

N

N NN

NH2

44

NON

N

N NN

NH2 I

12

N

N NN

NH2

45

N

23

argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP45. 1H NMR (CDCl3) δ 8.30 (s, 1H), 7.72 (d, J=7.9 Hz, 2H), 7.54 (d, J=7.7 Hz, 2H), 5.20 (m, J=6.7 Hz, 1H), 3.86 (s, 2H), 1.62 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 293.1, found 293.1; ESI-HRMS (M+H)+ m/z calcd 293.1515, found 293.1561.

Synthesis of 2-(3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)acetonitrile (PP46)

A solution of (3-Cyanomethylphenyl)boronic acid (18 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP46. 1H NMR (CDCl3) δ 8.24 (s, 1H), 7.70 (s, 1H), 7.6 (m, 2H), 7.55 (m, 2H), 5.21 (m, J=6.6 Hz, 1H), 3.88 (s, 2H), 1.63 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 293.1, found 293.1; ESI-HRMS (M+H)+ m/z calcd 293.1515, found 293.1523.

Synthesis of 1-isopropyl-3-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP48)

N

N NN

NH2 I

12

N

N NN

NH2

46

N

N

N NN

NH2 I

12

N

N NN

NH2

48

OMe

24

A solution of (4-Methoxyphenylboronic acid (17 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP48 (4.5mg, 16% yield). 1H NMR (CDCl3) δ 8.22 (s, 1H), 7.57 (d, J=8.5 Hz, 2H), 7.09 (d, J=8.7 Hz, 2H), 6.24 (br), 5.18 (m, J=6.6 Hz, 1H), 3.89 (s, 3H), 1.62 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 284.1, found 284.1; ESI-HRMS (M+H)+ m/z calcd 284.1512, found 284.1521.

Synthesis of 1-isopropyl-3-(3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP49)

A solution of 3-Methoxyphenylboronic acid (17 mg, 0.11 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.10 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP49. 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.49 (t, J=7.9 Hz, 1H), 7.19 (m, 1H), 7.17 (d, J=1.7 Hz, 1H), 7.08 (dd, J=8.4 Hz, J=2.4 Hz, 1H), 6.35 (br), 5.20 (m, J=6.6 Hz, 1H), 3.89 (s, 3H), 1.63 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 284.1, found 284.0; ESI-HRMS (M+H)+ m/z calcd 284.1515, found 284.1521.

Synthesis of 1-isopropyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP52)

N

N NN

NH2 I

12

N

N NN

NH2

49

OMe

N

N NN

NH2 I

12

N

N NN

NH2

N

52

25

A solution of 3-Pyridinylboronic acid (15 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (15 mg, 0.015 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O) to yield PP52. 1H NMR (CDCl3) δ 9.27 (s, 1H), 8.73 (d, J=3.85 Hz, 1H), 8.50 (d, J=7.7 Hz, 1H), 8.23 (s, 1H), 7.82 (t, J=5.8 Hz, 1H), 5.25 (m, J=6.9 Hz, 1H), 1.64 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 255.1, found 255.0; ESI-HRMS (M+H)+ m/z calcd 255.1358, found 255.1365.

Synthesis of 1-isopropyl-3-(pyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP53) A solution of 5-Pyrimidinylboronic acid (15 mg, 0.14 mmol) in EtOH (3.3 mL) was

added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (15 mg, 0.015 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O) to yield PP53. 1H NMR (CDCl3) δ 9.40 (s, 1H), 9.09 (s, 2H), 8.30 (s, 1H), 5.62 (m, J=6.3 Hz, 1H), 1.64 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 256.1, found 256.1; ESI-HRMS (M+H)+ m/z calcd 256.1311, found 256.1320.

N

N NN

NH2 I

12

N

N NN

NH2

NN

53

N

N NN

NH2 I

12

N

N NN

NH2

54

OO

26

Synthesis of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP54)

A solution of 2,3-dihydro-1,4-benzodioxin-6-ylboronic acid (26 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP54 (6 mg, 15% yield). 1H NMR (CDCl3) δ 8.21 (s, 1H), 7.13 (d, J=1.9 Hz, 1H), 7.07 (dd, J=8.2 Hz, J=1.9 Hz, 1H), 5.15 (m, J=7.0 Hz, 1H), 4.30 (s, 4H), 1.58 (d, J=7.2 Hz, 6H); ESI-MS (M+H)+ m/z calcd 312.1, found 312.0; ESI-HRMS (M+H)+ m/z calcd 312.1460, found 312.1470.

Synthesis of 1-(3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)ethanone (PP55)

A solution of 3-Acetylphenylboronic acid (23 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP55 (7 mg, 18% yield). 1H NMR (CDCl3) δ 8.26 (s, 2H-overlapping protons), 8.12 (d, J=7.4 Hz, 1H), 7.85 (d, J=7.1 Hz, 1H), 7.71 (t, J=7.8 Hz, 1H), 5.23 (m, J=6.6 Hz, 1H), 2.68 (s, 3H), 1.64 (d, J=6.65 Hz, 6H); ESI-MS (M+H)+ m/z calcd 296.1, found 296.1; ESI-HRMS (M+H)+ m/z calcd 296.1511, found 296.1520.

N

N NN

NH2 I

12

N

N NN

NH2

55

O

N

N NN

NH2 I

12

N

N NN

NH2

56

OH

27

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (PP56)

A solution of 4-Hydroxyphenylboronic acid (30 mg, 0.14 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.13 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP56 (12 mg, 32% yield). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.50 (m, J=8.7 Hz, 2H), 7.00 (m, J=8.5 Hz, 2H), 5.19 (m, J=6.6 Hz, 1H), 1.62 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 270.1, found 270.1; ESI-HRMS (M+H)+ m/z calcd 270.1355, found 270.1355.

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP59)

A solution of 3-fluoro-4-hydroxyphenylboronic acid (103 mg, 0.66 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP using silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP59 (26 mg, 27% yield). 1H NMR (CDCl3) δ 8.25 (s, 1H), 7.37 (dd, J=10.6 Hz, J=1.8 Hz, 7.26 (d, J=8.7 Hz, 1H), 7.14 (t, J=8.3 Hz, 1H), 5.12 (t, J=6.7 Hz, 1H) 1.54 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 288, found 288. ESI-HRMS (M+H)+ m/z calcd 288.12552, found 288.1256

N

N NN

NH2 I

12

N

N NN

NH2

59

OHF

28

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-3-methylphenol (PP60)

A solution of 4-hydroxy-2-methylphenylboronic acid (110 mg, 0.66 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP60 (42 mg, 22% yield). 1H NMR (CDCl3) δ 8.34 (s, 1H), 7,23 (s, 1H), 6.86 (d, J=2.3 Hz, 1H), 6.81 (dd, J=8.2 Hz, J=2.8 Hz, 1H), 5.18 (m, J=6.6 Hz, 1H), 2.26 (s, 3H), 2.4 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 284, found 284. ESI-HRMS (M+H)+ m/z calcd 288.15059, found 284.1506.

Synthesis of 3-(4-fluoro-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP62)

A solution of 4-fluoro-3-methoxyphenylboronic acid (61 mg, 0.36 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (90 mg, 0.30 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP62 (40 mg, 44% yield). 1H NMR (CDCl3) δ 8.33 (s, 1H), 7.27 (dd, J=8.1 Hz, J=2.3 Hz, 1H), 7.21 (d, J=10.6 Hz, 1H), 7.17 (dd, J=4.4 Hz, J=1.8 Hz, 1H), 5.17 (m, J=6.5 Hz, 1H), 3.95 (s, 3H); ESI-MS (M+H)+ m/z calcd 302, found 302. ESI-HRMS (M+H)+ m/z calcd 302.14117, found 302.1412.

N

N NN

NH2 I

12

N

N NN

NH2

60

OH

Me

N

N NN

NH2 I

12

N

N NN

NH2

62

F

OMe

29

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP262) A solution of PP62 (3-(4-fluoro-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, 30 mg, 0.10 mmol) was dissolved in CH2Cl2 (5 mL) and stirred under an argon atmosphere. BBr3 (500uL, 0.5 mol) was added slowly with a syringe, while stirring. The reaction was stirred at room temperature for 3 hours then concentrated in vacuo and purified using silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP262 (23 mg, 44% yield). 1H NMR (CDCl3) δ 8.34 (s, 1H), 7.35 (dd, J=8.1 Hz, J=1.5 Hz, 1H), 7.21 (d, J=10.3 Hz, 1H), 7.18 (dd, J=4.4 Hz, 1.4 Hz, 1H), 5.18 (m, J=6.5 Hz, 1H), 1.59 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 288.1, found 288.1. ESI-HRMS (M+H)+ m/z calcd 288.12552, found 288.1255.

Synthesis of 3-(2,5-difluoro-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP63)

A solution of 2,5-difluoro-4-methoxyphenylboronic acid (84 mg, 0.45 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (54 mg, 0.18 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP63 (50 mg, 17% yield). 1H NMR (CDCl3) δ 8.35 (s, 1H), 7.35 (dd, J=11.0 Hz, J=6.9, 1H), 6.87 (dd, J=11.0 Hz, J=7.0, 1H), 5.18 (m, J=6.5 Hz, 1H), 3.95 (s, 3H), 1.59 (d, J=6.8 Hz, 6H); ESI-MS (M+H)+ m/z calcd 320.1, found 320.0. ESI-HRMS (M+H)+ m/z calcd 320.13174, found 320.1324.

N

N NN

NH2 I

12

N

N NN

NH2

63

OMeF

F

N

N NN

NH2

62

F

OMe

N

N NN

NH2

F

OH

262

30

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2,5-difluorophenol (PP93)

3-(2,5-difluoro-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA63) (20 mg, 0.06 mmol) was dissolved in CH2Cl2 (2mL) and BBr3 (0.630 mL, 0.63 mmol) was added slowly with a syringe, while stirring. The reaction was stirred at room temperature for overnight then concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP93 (6.7 mg, 35% yield). 1H NMR (CDCl3) δ 8.27 (s, 1H), 7.37 (dd, J=10.4 Hz, J=6.7 Hz, 1H), 7.00 (dd, J=10.6 Hz, J=7.0 Hz, 1H), 5.21 (m, J=6.5 Hz, 1H), 1.62 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 306.1, found 306.0. ESI-HRMS (M+H)+ m/z calcd 306.11609, found 306.1162.

Synthesis of 1-isopropyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP64)

A solution of 3,4,5-trimethoxyphenylboronic acid (123 mg, 0.58 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (70 mg, 0.23 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP64 (70 mg, 89% yield). 1H NMR (CDCl3) δ 8.34 (s, 1H), 6.88 (s, 2H), 5.16 (m, J=6.7 Hz, 1H), 3.91 (s, 6H), 3.88 (s, 3H), 1.59 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 344.1, found 344.0. ESI-HRMS (M+H)+ m/z calcd 344.17172, found 344.1726.

N

N NN

NH2 I

12

N

N NN

NH2

93

OHF

F

N

N NN

NH2 I

12

N

N NN

NH2

64

OMeMeOOMe

31

Synthesis of 1-isopropyl-3-(2,3-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP65)

A solution 2,3-dimethoxyphenylboronic acid (105 mg, 0.58 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (70 mg, 0.23 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP65 (63 mg, 88% yield). 1H NMR (CDCl3) δ 8.31 (s, 1H), 7.18 (d, J=7.6 Hz, 1H), 7.14 (dd, J=7.8 Hz, J=1.9 Hz, 1H), 7.02 (dd, J=8.1 Hz, J=1.8 Hz, 1H), 5.17 (m, J=6.5 Hz, 1H), 3.92 (s, 3H), 3.68 (s, 3H), 1.59 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 314.1, found 314.1. ESI-HRMS (M+H)+ m/z calcd 314.16115, found 314.1613.

Synthesis of 1-isopropyl-3-(2,4-dimethoxypyrimidin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP66)

A solution 2,4-dimethoxypyrimidin-5-yl-5-boronic acid (106 mg, 0.58 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (70 mg, 0.23 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by silica gel column chromatography [MeOH—CH2Cl2, 2:98] to yield PP66. 1H NMR (CDCl3) δ 8.46 (s, 1H), 8.32 (s, 1H), 5.16 (m, J=6.5 Hz, 1H), 4.06 (s, 3H), 4.05 (s, 3H), 1.57 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 316.1, found 316.0. ESI-HRMS (M+H)+ m/z calcd 316.15165, found 316.1517.

N

N NN

NH2 I

12

N

N NN

NH2

65

OMe

OMe

N

N NN

NH2 I

12

N

N NN

NH2

66

NN

OMe

OMe

32

OMe

OHO

OMe

ONC

CN

OMe

OMeNC

CN

NH

NNC

H2N

N

N NH

N

NH2

OMe

OCl

OMe OMe

68 7270

76 74

F F F F

FF

Synthesis of 3-(2-fluoro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA76)

2-fluoro-3-methoxybenzoic acid (2 g, 0.012 mol) was stirred in CH2Cl2 (20mL) at 0°C under an argon atmosphere. DMF (5 drops, catalytic) was added, followed by oxalyl chloride (5.09 mL, 0.059 mol). Reaction was warmed to room temperature then stirred under argon for one hour. Reaction was concentrated in vacuo to yield 2-fluoro-3-methoxybenzoyl chloride (BA68). A solution of malononitrile (1.19 g, 0.018 mol) in dry THF (2 mL) was stirred at 0°C under an argon atmosphere. NaH in paraffin oil (1.92 g, 0.048 mol) was added piece-wise to solution. 2-fluoro-3-methoxybenzoyl chloride (BA68, 0.012 mol) was dissolved in 20 mL dry THF and added slowly to reaction. Reaction was warmed to room temperature and stirred under argon for 24 hours. 1N HCl (40 mL) was slowly added, then reaction mixture was extracted with EtOAc. Organic phases were combined, dried with magnesium sulfate, then concentrated in vacuo to yield 2-((2-fluoro-3-methoxyphenyl)(hydroxy)methylene)malononitrile (BA70).

2-((2-fluoro-3-methoxyphenyl)(hydroxy)methylene)malononitrile (BA70, 0.012 mol) and sodium bicarbonate (8.06 g, 96 mmol) were combined in a solution of 1,4-dioxane (32 mL) and water (5.3 mL). Dimethylsulphate (7.99 mL, 84 mmol) was slowly added and the reaction was heated to 80-90º C for 2 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with diethyl ether (100 mL). The organic phases were combined, dried with MgSO4, and filtered to give 2-((2-fluoro-3-methoxyphenyl)(methoxy)methylene)malononitrile (BA72), which appeared as an orange oil and was purified by silica gel chromatography (10% EtOAc/Hexanes slowly increasing to 40% EtOAc/Hexanes) to give a yellow oil.

2-((2-fluoro-3-methoxyphenyl)(methoxy)methylene)malononitrile (BA72, 0.012 mmol) was stirred in EtOH (20mL) at room temperature under an argon atmosphere. Hydrazine (0,58 mL, 12 mmol) was added and reaction was left stirring for 90 minutes. Reaction mixture was concentrated in vacuo and dried on vacuum pump overnight to yield intermediate 5-amino-3-(2-fluoro-3-methoxyphenyl)-1H-pyrazole-4-carbonitrile

33

(BA74), which appeared as an orange oil and was taken on without further characterization.

Formamide (20mL) was added to 5-amino-3-(2-fluoro-3-methoxyphenyl)-1H-pyrazole-4-carbonitrile (BA74, 0.012 mol) and the reaction was heated to 180°C under an argon atmosphere overnight. Reaction was cooled and dH2O was added (40mL) forcing a white precipitate out of solution. Precipitate was collected and washed with dH2O. Solid was dried and purified by silica gel column chromatography [MeOH—CH2Cl2, 10:90] to yield 3-(2-fluoro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA76). ESI-MS (M+H)+ m/z calcd 259.1, found 260.11.

N

N NH

N

NH2

OMe

N

N NN

NH2

OMe

BA7776

F FN

N NN

NH2

OH

PP77

F

Synthesis of 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP77) 3-(2-fluoro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA76, 100 mg, 0.386 mmol) was dissolved in DMF (2 mL). K2CO3 (213 mg, 1.54 mmol) was added and reaction was stirred at room temperature under an argon atmosphere. 2-iodopropane (0.113 mL, 1.16 mmol) was added with a syringe and reaction was stirred for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Water (50 mL) was added and reaction was extracted with CH2Cl2. Organic phases were dried with sodium sulfate, concentrated in vacuo yielding an oil. The oil was dissolved in 10 mL CH2Cl2 and placed under argon. BBr3 (3.84 mL, excess) was added and reaction was stirred for 45 minutes at room temperature. The reaction was quenched with saturated sodium bicarbonate (aqueous) and extracted three times with CH2Cl2 (20 mL). Organic phases were combined, concentrated in vacuo and the final product was purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid, PP77 (56 mg, 51% yield). 1H NMR (CDCl3) δ 8.24 (s, 1H), 7.20 (m, J=1.96 Hz, 1H), 7.18 (m, 1H), 7.07 (m, J=2.1 Hz, 1H), 6.32 (br), 5.21 (m, J=6.7 Hz, 1H), 1.62 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 287.1, found 288.11. ESI-HRMS (M+H)+ m/z calcd 288.12552, found 288.1256.

34

N

N NH

N

NH2

OMe

N

N NN

NH2

OMe

BA7876

F FN

N NN

NH2

OH

PP78

F

Synthesis of 3-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP78) 3-(2-fluoro-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA76, 100 mg, 0.386 mmol) was dissolved in DMF (2 mL). K2CO3 (250 mg) was added and reaction was stirred at room temperature under an argon atmosphere. Iodocyclopentane (0.066 mL, 1.5 mmol) was added with a syringe and reaction was stirred for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Water (50 mL) was added and reaction was extracted with CH2Cl2. Organic phases were dried with sodium sulfate, concentrated in vacuo yielding an oil. The oil was dissolved in 10 mL CH2Cl2 and placed under argon. BBr3 (3.84 mL, excess) was added and reaction was stirred for 45 minutes at room temperature. The reaction was quenched with saturated sodium bicarbonate (aqueous) and extracted three times with CH2Cl2 (20 mL). Organic phases were combined, concentrated in vacuo and the final product was purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid, PP78 (12 mg, 10% yield). 1H NMR (CDCl3) δ 8.26 (s, 1H), 7.91 (m, 1H), 7.21 (m, 1H), 7.07 m, 1H), 5.34 (m, J=7.7 Hz, 1H), 2.19 (m, 4H), 2.01 (m, 2H), 1.77 (m, 2H); ESI-MS (M+H)+ m/z calcd 313.1 found 314.1. ESI-HRMS (M+H)+ m/z calcd 314.14117, found 314.1421.

35

OMe

OHO

OMe

ONC

CN

OMe

OMeNC

CN

NH

NNC

H2N

N

N NH

N

NH2

F OMe

OCl

F F F

FOMe

FOMe

N

N NN

NH2

FOMe

79

67 7169

75 73 Synthesis of 1-cyclopentyl-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA79)

3-fluoro-5-methoxybenzoic acid (5 g, 0.029 mol) was stirred in CH2Cl2 (50mL) at 0°C under an argon atmosphere. DMF (9 drops, catalytic) was added, followed by oxalyl chloride (12.7 mL, 0.147 mol). Reaction was warmed to room temperature then stirred under argon for one hour. Reaction was concentrated in vacuo to yield 3-fluoro-5-methoxybenzoyl chloride (BA67). A solution of malononitrile (2.87 g, 0.044 mol) in dry THF (50 mL) was stirred at 0°C under an argon atmosphere. NaH in paraffin oil (4.64 g, 0.116 mol) was added piece-wise to solution. 3-fluoro-5-methoxybenzoyl chloride (BA67, 0.029 mol) was dissolved in 50 mL dry THF and added slowly to reaction. Reaction was warmed to room temperature and stirred under argon for 24 hours. 1N HCl (200 mL) was slowly added, then reaction mixture was extracted with EtOAc. Organic phases were combined, dried with magnesium sulfate, then concentrated in vacuo to yield 2-((3-fluoro-5-methoxyphenyl)(hydroxy)methylene)malononitrile (BA69).

2-((3-fluoro-5-methoxyphenyl)(hydroxy)methylene)malononitrile (BA69, 29 mmol)and sodium bicarbonate (19.5 g, 232 mmol) were combined in a solution of 1,4-dioxane (50 mL) and water (10 mL). Dimethylsulphate (19.3 mL, 203 mmol) was slowly added and the reaction was heated to 80-90º C for 2 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with diethyl ether (200 mL). The organic phases were combined, dried with MgSO4, and filtered to give 2-((3-fluoro-5-methoxyphenyl)(methoxy)methylene)malononitrile (BA71), which appeared as a brown oil and was purified using silica gel chromatography (10% EtOAc/Hexanes slowly increasing to 40% EtOAc/Hexanes) to give a yellow oil.

2-((3-fluoro-5-methoxyphenyl)(methoxy)methylene)malononitrile (BA71, 29mmol) was stirred in EtOH (20mL) at room temperature under an argon atmosphere. Hydrazine (1.4 mL, 29 mmol) was added and reaction was left stirring for 90 minutes. Reaction mixture was concentrated in vacuo and dried on vacuum pump overnight to yield intermediate 5-amino-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazole-4-carbonitrile

36

(BA73). Formamide (20mL) was added and reaction was heated to 180°C under an argon atmosphere overnight. Reaction was cooled and dH2O was added (40mL) forcing a white precipitate out of solution. Precipitate was collected and washed with dH2O. Solid was dried and purified by silica gel column chromatography [MeOH—CH2Cl2, 10:90] to yield 3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA75).

3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA75, 100 mg, 0.386 mmol) was dissolved in DMF (10 mL). K2CO3 (250 mg, 1.54 mmol) was added and reaction was stirred at room temperature under an argon atmosphere. Iodocyclopentane (0.134 mL, 1.16 mmol) was added with a syringe and reaction was stirred for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Sodium citrate (50 mL) was added and reaction was extracted with EtOAc. Organic phases concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield BA79.

Synthesis of 1-cyclopentyl-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP79) 1-cyclopentyl-3-(3-fluoro-5-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA79, 0.386 mmol) was dissolved in CH2Cl2 (2 mL). BBr3 (4 mL, 4 mol) was added slowly with a syringe, while stirring. The reaction was stirred at room temperature for 2 hours then concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield BA79 (69 mg, 57% yield). 1H NMR (CDCl3) δ 8.25 (s, 1H), 7.07 (s, 1H), 6.98 (d, J=8.1 Hz, 1H), 6.75 (dt, J=9.9 Hz, J=2.2 Hz, 1H), 5.32 (m, J=7.3 Hz, 1H), 2.18 (m, 4H), 2.02 (m, 2H), 1.78 (m, 2H). ESI-HRMS (M+H)+ m/z calcd 314.14117, found 314.1422.

Synthesis of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA80)

A solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (400 mg, 1.53 mmol) and K2CO3 (1 g, 6 mmol) in DMF (5 mL) was stirred at room temperature under an argon

N

N NNH

NH2 I

N

N NN

NH2 I

19 80

N

N NN

NH2

FOMe

BA79

N

N NN

NH2

FOH

PP79

37

atomosphere. Iodocyclopentane (1.0g, 0.0084 mol) was added with a syringe. Reaction was refluxed under argon atmosphere for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Sodium citrate (50 mL) was added and reaction was extracted with EtOAc. Organic phases concentrated in vacuo and purified using silica gel column chromatography [MeOH—CH2Cl2, 5:95] yielding B80 (300 mg, 60% yield). ESI-MS (M+H)+ m/z calcd 330.0, found 330.0.

N

N NN

NH2 I

BA80

N

N NN

NH2

BA81

MeO HN O

O

N

N NN

NH2

PP81

MeO NH2

N

N NN

NH2

PP281

HO NH2

Synthesis of tert-butyl 4-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylcarbamate (BA81), 3-(4-amino-3-methoxyphenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP81), 2-amino-5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (PP281) A solution of tert-butyl 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylcarbamate (200 mg, 0.76 mmol) in EtOH (3.3 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA80, 100 mg, 0.30 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified using silica gel column chromatography [MeOH—CH2Cl2, 5:95] yielding tert-butyl 4-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylcarbamate (BA81). BA81 was dissolved in 50:50 CH2Cl2:TFA and stirred for one hour at room temperature. The reaction mixture was concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP81. ESI-HRMS (M+H)+ m/z calcd 325.17714, found 325.1772. PP81 was dissolved in CH2Cl2 (2mL) and BBr3 (4 mL, 4 mol) was added slowly with a syringe, while stirring. The reaction was stirred at room temperature for 2 hours then concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP281. ESI-HRMS (M+H)+ m/z calcd 311.16149, found 311.1616

38

Synthesis of 3-(3-bromo-5-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP85)

A solution of 2-(3-bromo-5-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (137 mg, 0.43 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (65 mg, 0.216 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP85 (28mg, 36% yield). 1H NMR (CDCl3) δ 8.25 (s, 1H), 7.38 (t, J=1.5 Hz, 1H), 7.20 (t, J=2.1 Hz, 1H), 7.09 (m, 1H), 5.19 (m, J=6.7 Hz, 1H), 3.87 (s, 3H), 1.61 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 362.1, found 362.0. ESI-HRMS (M+H)+ m/z calcd 362.0611, found 362.0611. Synthesis of 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-5-bromophenol (PP87)

3-(3-bromo-5-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP85, 0.1 mmol) was dissolved in CH2Cl2 (1 mL) and BBr3 (1 mL, 1 mol) was added slowly with a syringe, while stirring. The reaction was stirred at room temperature for 35 minutes then concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP87 (10.7 mg, 31% yield). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.33 (m, 1H), 7.20 (m, 1H), 7.13 (m, 1H), 5.18 (m, J=7.1 Hz, 1H), 1.61 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 348.0, found 348.0. ESI-HRMS (M+H)+ m/z calcd 348.04545, found 348.0455

N

N NN

NH2 I

12

N

N NN

NH2

86

N

OO

N

N NN

NH2

HN

89

N

N NN

NH2 I

12

N

N NN

NH2

BrOMe

N

N NN

NH2

BrOH

85 87

39

Synthesis of tert-butyl 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-1-carboxylate (PP86)

A solution of tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate (212 mg, 0.61 mmol) in EtOH (3.3 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (75 mg, 0.25 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O) to yield PP86 (9.3 mg, 9% yield). 1H NMR (CD3OD) δ 8.34 (s, 1H), 8.30 (d, J=8.4 Hz, 1H), 7.89 (d, J=1.7 Hz, 1H), 7.68 (d, J=3.7 Hz, 1H), 7.62 (dd, J=8.5 Hz, J=2.0 Hz, 1H), 6.65 (d, J=3.5 Hz, 1H), 5.20 (m, J=7.1 Hz, 1H), 1.70 (s, 9H), 1.62 (d, J=7.0 Hz, 6H). ESI-HRMS (M+H)+ m/z calcd 393.20335, found 393.2035. Synthesis of 3-(1H-indol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP89)

Tert-butyl 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-1-carboxylate (PP86, 9 mg, 0.022 mmol) was dissolved in 50:50 CH2Cl2:TFA and stirred for one hour at room temperature. The reaction mixture was concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP89 (4.8 mg, 75% yield). 1H NMR (CDCl3) δ 8.56 (br), 8.21 (s, 1H), 7.96 (s, 1H), 7.58 (d, J=7.6 Hz, 1H), 7.43 (dd, J=8.1 Hz, J=1.4 Hz, 1H), 7.34 (t, J=2.6 Hz, 1H), 6.65 (m, 1H), 6.42 (br), 5.20 (m, J=6.7 Hz, 1H), 1.64 (d, J=6.8 Hz, 1H); ESI-MS (M+H)+ m/z calcd 293.1, found 293.0. ESI-HRMS (M+H)+ m/z calcd 293.15092, found 293.1509

Synthesis of tert-butyl 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-1-carboxylate (PP88)

A solution of tert-butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-1-carboxylate (130 mg, 0.38 mmol) in EtOH (3.3 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) in DME (12 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (1.9 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O) to yield PP88. 1H NMR (CD3OD) δ 8.36 (d, J=6.6 Hz, 1H), 8.35 (s, 1H), 7.93 (d, J=1.9 Hz, 1H), 7.77 (d, J=3.7 Hz, 1H), 7.65 (dd, J=8.3 Hz, J=1.9 Hz, 1H), 6.77 (d, J=3.7 Hz, 1H), 5.38 (m, J=7.4 Hz, 1H), 2.22 (m, 4H), 2.05 (m, 2H), 1.82 (m, 2H); ESI-MS (M+H)+ m/z calcd 419.2, found 419.1. ESI-HRMS (M+H)+ m/z calcd 419.219, found 419.219.

N

N NN

NH2 I

80

N

N NN

NH2

88

N

OO

N

N NN

NH2

HN

94

40

Synthesis of 3-(1H-indol-5-yl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP94)

Tert-butyl 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-1-carboxylate (PP88) was dissolved in 50:50 CH2Cl2:TFA and stirred for one hour at room temperature. The reaction mixture was concentrated in vacuo and purified using by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP94 (6.3 mg). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.90 (s, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.43 (dd, J=8.4 Hz, J=1.5 Hz, 1H), 7.35 (t, J=2.9 Hz, 1H), 6.65 (m, 1H), 5.33 (m, J=7.7 Hz, 1H), 2.22 (m, 4H), 2.03 (m, 2H), 1.77 (m, 2H); ESI-MS (M+H)+ m/z calcd 319.1, found 319.2. ESI-HRMS (M+H)+ m/z calcd 319.16657, found 319.1667.

Synthesis of 1-cyclopentyl-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP90)

A solution of 3,4-dimethoxyphenylboronic acid (41 mg, 0.23 mmol) in EtOH (1.65mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.09 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O) to yield PP90 (8.4 mg, 28% yield). 1H NMR (CDCl3) δ 8.22 (s, 1H), 7.15 (dd, J=7.0 Hz, J=2.0 Hz, 1H), 7.14 (s, 1H), 7.03 (d, J=8.8 Hz, 1H), 5.31 (m, J=7.6 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 2.20 (m, 4H), 2.02 (m, 2H), 1.77 (m, 2H); ESI-MS (M+H)+ m/z calcd 340.2, found 340.1. ESI-HRMS (M+H)+ m/z calcd 340.1768, found 340.1768.

Synthesis of 3-(1H-indol-4-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP91)

A solution of 1H-indol-4-yl-4-boronic acid (40 mg, 0.25 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30

N

N NN

NH2 I

80

N

N NN

NH2

90

OMe

OMe

N

N NN

NH2 I

12

N

N NN

NH2

91

NH

41

mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP91 (14.6 mg, 50% yield). 1H NMR (CDCl3) δ 8.76 (br, 1H), 8.24 (s, 1H), 7.58 (d, J=7.3 Hz, 1H), 7.36 (m, 1H), 7.35 (m, 1H), 7.34 (s, 1H), 6.57 (s, 1H), 5.25 (m, J=6.9 Hz, 1H), 1.67 (d, J=6.5 Hz, 6H); ESI-MS (M+H)+ m/z calcd 293.1, found 293.1. ESI-HRMS (M+H)+ m/z calcd 293.15092, found 293.1511.

Synthesis of 1-cyclopentyl-3-(1H-indol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP92)

A solution of 1H-indol-4-yl-4-boronic acid (30 mg, 0.19 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.076 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP92 (23 mg, 95% yield). 1H NMR (CDCl3) δ 8.74 (br, 1H), 8.23 (s, 1H), 7.58 (d, J=7.3 Hz, 1H), 7.37 (m, 1H), 7.35 (m, 1H), 7.33 (m, 1H), 6.57 (m, 1H), 5.38 (m, J=7.4 Hz, 1H), 2.25 (m, 4H), 2.03 (m, 2H), 1.79 (m, 2H); ESI-MS (M+H)+ m/z calcd 319.2, found 319.1. ESI-HRMS (M+H)+ m/z calcd 319.16657, found 319.1667.

Synthesis of 3-(2,3-dihydrobenzofuran-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP95)

A solution of 2,3-dihydro-1-benzofuran-5-ylboronic acid (38 mg, 0.23 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 80°C under

N

N NN

NH2 I

80

N

N NN

NH2

92

NH

N

N NN

NH2 I

12

N

N NN

NH2

95

O

42

an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP95 (15.7 mg, 59% yield). 1H NMR (CDCl3) δ 11.68 (br), 8.21 (s, 1H), 7.49 (s, 1H), 7.37 (d, J=8.2 Hz, 1H), 6.95 (d, J=8.2 Hz, 1H), 5.18 (m, J=7.0 Hz, 1H), 4.69 (t, J=8.6 Hz, 2H), 3.31 (t, J=8.6 Hz, 2H), 1.62 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 296.1, found 296.1. ESI-HRMS (M+H)+ m/z calcd 296.15059, found 296.1507.

Synthesis of 3-(benzofuran-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP96)

A solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-benzofuran (56 mg, 0.23 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP96 (19 mg, 72% yield). 1H NMR (CDCl3) δ 8.24 (s, 1H), 7.89 (d, J=1.5 Hz, 1H), 7.75 (d, J=2.2 Hz, 1H), 7.69 (d, J=8.8 Hz, 1H), 7.57 (dd, J=8.4 Hz, J=1.8 Hz, 1H), 6.87 (dd, J=2.2 Hz, J=0.7 Hz, 1H), 5.20 (m, J=7.0 Hz, 1H), 1.63 (d, J=7.0 Hz, 6H). ESI-HRMS (M+H)+ m/z calcd 294.13494, found 294.1351.

N

N NN

NH2 I

80

N

N NN

NH2

97

O

Synthesis of 3-(benzofuran-5-yl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP97)

5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-benzofuran (37 mg, 0,071 mmol) ) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.076 mmol) in DME (6 mL). Pd(PPh3)4 (30 mg, 0.03 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was

N

N NN

NH2 I

12

N

N NN

NH2

96

O

43

heated to 80°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP97 (16 mg, 83% yield). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.88 (s, 1H), 7.77 (d, J=2.2 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.56 (dd, J=8.4 Hz, J=1.8 Hz, 1H), 6.88 (m, 1H), 5.34 (m, J=7.3 Hz, 1H), 2.21 (m, 4H), 2.02 (m, 2H), 1.78 (m, 2H) ; ESI-MS (M+H)+ m/z calcd 320.1, found 320.0. ESI-HRMS (M+H)+ m/z calcd 320.15059, found 320.1508.

Synthesis of 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-ethoxyphenol (PP98)

1-cyclopentyl-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP359, 25 mg, 0,071 mmol) was dissolved in CH2Cl2 (5 mL) and stirred at -10°C under an argon atmosphere. After 30 minutes, reaction was brought to 0°C and stirred for 2.5 hours. Reaction was stirred for additional 4 hours at room temperature, then concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP98 (3 mg, 13% yield). 1H NMR (CDCl3) δ 7.23 (d, J=2.2 Hz, 1H), 7.07 (dd, J=8.1 Hz, J=2.2 Hz, 1H), 5.31 (m, J=7.3 Hz, 1H), 4.22 (m, 2H), 2.33 (m, 4H), 1.99 (m, 2H), 1.75 (m, 2H), 1.51 (m, 3H); ESI-MS (M+H)+ m/z calcd 340.1, found 340.1. ESI-HRMS (M+H)+ m/z calcd 340.1768, found 340.1768.

N

N NN

NH2

359

O

O

N

N NN

NH2

98

O

OH

N

N NN

NH2

NH2

OMe

20d

N

N NN

NH2

HN

OMe

OH

99

44

Synthesis of 2-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenylamino)propan-1-ol (PP99)

3-(4-amino-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (pp202) (30 mg, 0.10 mmol) was dissolved in DMF (0.400 mL). K2CO3 (55 mg, 0.4 mmol) was added and reaction was stirred at 70°C. 3-bromo-1-propanol (0.050 mL, 0.6 mmol) was added and reaction was stirred overnight. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Sodium citrate (50 mL) was added and reaction was extracted with saturated NaCl and CH2Cl2. Organic phases concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP99 (8.4 mg, 24% yield). ESI-MS (M+H)+ m/z calcd 357.2, found 357.1. ESI-HRMS (M+H)+ m/z calcd 357.20335, found 357.2034.

Synthesis of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA109). A solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2 g, 7.69 mmol) and K2CO3 (4.25 g, 30.8 mmol) in DMF (5 mL) was stirred at room temperature under an argon atomosphere. Iodomethane (1.17 mL, 7.69 mmol) was added with a syringe. Reaction was stirred under an argon atmosphere at room temperature for 2 hours. Solid K2CO3 was removed by filtration. Solvent was partially removed in vacuo. Sodium citrate (50 mL) was added and reaction was extracted with EtOAc. Organic phases concentrated in vacuo and purified using silica gel column chromatography [MeOH—CH2Cl2, 5:95] yielding BA109 (212 mg, 10% yield). 1H NMR (CDCl3) δ 8.31 (s, 1H), 4.07 (s, 3H); ESI-MS (M+H)+ m/z calcd 275.9, found 275.9.

Synthesis of 1-isopropyl-3-(quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP102) A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (63 mg, 0.25 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP102 (27.5 mg, 19 % yield). 1H NMR (CDCl3) δ 9.0 (dd, J=1.5 Hz, J=4.1 Hz, 1H), 8.42 (s, 1H), 8.29 (s, 1H),

N

N NNH

NH2 I

19

N

N NN

NH2 I

109

N

N NN

NH2 I

12

N

N NN

NH2

102

N

45

8.27 (d, J=7.3 Hz, , 1H), 8.18 (d, J=2.0 Hz, 1H), 8.09 (dd, J=2.1 Hz, J=8.7 Hz, 1H), 7.50 (dd, J=4.3 Hz, J=8.3 Hz, 1H), 5.44 (br, 2H), 5.24 (m, J=6.4 Hz, 1H), 1.64 (d, J=6.7 Hz, 6H); ESI-MS (M+H)+ m/z calcd 305.14, found 305.10. ESI-HRMS (M+H)+ m/z calcd 305.1515, found 305.1504.

Synthesis of 1-isopropyl-3-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP105) A solution of quinolin-3-yl-3-boronic acid (43 mg, 0.25 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 1mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP105 (45 mg, 85% yield). 1H NMR (CDCl3) δ 9.24 (d, J=2.2 Hz, 1H), 8.44 (d, J=2.2 Hz, 1H), 8.34 (s, 1H), 8.15 (d, J=8.4 Hz, 1H), 7.90 (d, J=8.1 Hz, 1H), 7.79 (t, J=7.0 Hz, 1H), 7.63 (t, J=7.7 Hz, 1H), 5.23 (m, J=6.6 Hz, 1H), 1. 3 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 305.14, found 305.10. ESI-HRMS (M+H)+ m/z calcd 305.15092, found 305.151.

Synthesis of 3-(1H-indol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP107) A solution of indole-6-boronic acid (40.25 mg, 0.25 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP107 (22 mg, 42% yield). 1H NMR (CDCl3) δ 8.30 (s,

N

N NN

NH2 I

12

N

N NN

NH2

N

105

N

N NN

NH2 I

12

N

N NN

NH2

107

NH

46

1H), 7.84 (d, J=8.1 Hz, 1H), 7.72 (s, 1H), 7.39 (m, 1H), 7.37 (d, J=2.9 Hz, 1H), 6.66 (m, 1H), 5.22 (m, J=6.6 Hz, 1H), 1.65 (d, J=6.6 Hz, 1H); ESI-MS (M+H)+ m/z calcd 293.14, found 293.10. ESI-HRMS (M+H)+ m/z calcd 293.15092, found 293.151.

Synthesis of 1-isopropyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP108) A solution of 7-azaindole-5-boronic acid (61 mg, 0.25 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.1 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP108 (34 mg, 65% yield). 1H NMR (CDCl3) δ 8.66 (s, 2H, overlapping singlets), 8.25 (s, 1H), 7.60 (d, J=2.6 Hz, 1H), 6.81 (d, J=2.6 Hz, 1H), 5.25 (m, J=7.0 Hz, 1H), 1.64 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 294.14, found 294.10. ESI-HRMS (M+H)+ m/z calcd 294.14617, found 294.1462.

Synthesis of 3-(1H-indazol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP110) A solution of 6-indazolboronic acid (30 mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (22 mg, 0.074 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP110 (15.5 mg, 40% yield). 1H NMR (CDCl3) δ 8.28 (s, 1H), 8.22 (s, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.87 (s, 1H), 7.47 (d, J=8.1 Hz, 1H), 5.24 (m,

N

N NN

NH2 I

12

N

N NN

NH2

N

108

HN

N

N NN

NH2 I

12

N

N NN

NH2

110

NHN

47

J=6.6 Hz, 1H), 6.65 (d, J=7.0 Hz, 6H), ESI-MS (M+H)+ m/z calcd 294.14, found 294.10. ESI-HRMS (M+H)+ m/z calcd 294.14617, found 294.1462.

Synthesis of 1-isopropyl-3-(2-methylquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP118) A solution of 2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (ZK507, 10 mg, 0.04 mmol) in EtOH (0.8 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5 mg, 0.015 mmol) in DME (3 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.5 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP118 (8 mg, 97% yield). 1H NMR (CDCl3) δ 8.76 (m, 2H), 8.32 (m, 1H), 8.30 (s, 1H), 7.72 (m, 1H), 7.26 (s, 2H), 5.26 (m J=6.9 Hz, 1H), 3.11 (s, 3H), 1.65 (d, J=6.9 Hz, 6H); ESI-MS (M+H)+ m/z calcd 319.16, found 319.10. ESI-HRMS (M+H)+ m/z calcd 319.16657, found 319.1667.

Synthesis of 1-isopropyl-3-(isoquinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP129) A solution of isoquinolin-6-yl-6-boronic acid (PP127, 29 mg, 0.165 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP129 (30 mg, 34% yield). ESI-HRMS (M+H)+ m/z calcd 305.15092, found 305.151.

N

N NN

NH2 I

12

N

N NN

NH2

118

NMe

N

N NN

NH2 I

12

N

N NN

NH2

129

N

48

Synthesis of 3-(2-chloroquinolin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP130) A solution of 2-chloroquinolin-6-yl-6-boronic acid (ZK526, 60 mg, 0.28 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP130 (11 mg, 28% yield). 1H NMR (CDCl3) δ 8.28 (s, 1H), 8.25 (d, J=8.4 Hz, 1H), 8.22 (d, J=8.8 Hz, 1H), 8.12 (d, J=1.8 Hz, 1H), 8.05 (dd, J=8.8 Hz, J=1.8 Hz, 1H), 7.54 (d, J=8.8 Hz, 1H), 5.26 (m, J=6.6 Hz, 1H), 1.66 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 339.1, found 339.0. ESI-HRMS (M+H)+ m/z calcd 339.11195, found 339.112.

Synthesis of 1-isopropyl-3-(isoquinolin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP131) A solution of isoquinolin-7-yl-7-boronic acid (ZK528, 48 mg, 0.28 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP131 (14 mg, 38% yield). 1H NMR (CDCl3) δ 8.78 (s, 1H), 8.57 (d, J=8.4 Hz, 1H), 8.48 (d, J=8.4 Hz, 1H), 8.28-8.25 (m, 3H (overlapping spectra)), 8.20 (d, J=5.5 Hz, 1H), 5.28 (m, J=6.6 Hz, 1H), 1.66 (d, J=6.6 Hz, 6H). ESI-HRMS (M+H)+ m/z calcd 305.15092, found 305.1511.

N

N NN

NH2 I

12

N

N NN

NH2

130

NCl

N

N NN

NH2 I

12

N

N NN

NH2

131

N

49

Synthesis of 1-isopropyl-3-(quinoxalin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP132) A solution of quinoxalin-6-yl-6-boronic acid (ZK515, 30 mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP132 (23 mg, 62% yield). 1H NMR (CDCl3) δ 8.95 (m, 2H), 8.40 (d, J=2.2 Hz, 1H), 8.34 (d, J=8.4 Hz, 1H), 8.28 (s, 1H), 8.12 (dd, J=8.8 Hz, J=1.8 Hz, 1H), 5.27 (m, J=6.6 Hz, 1H), 1.67 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 306.14, found 306.10. ESI-HRMS (M+H)+ m/z calcd 306.14617, found 306.1463.

Synthesis of 1-isopropyl-3-(2-methylquinolin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP133) A solution of 2-methylquinolin-7-yl-7-boronic acid (ZK531, 40 mg, 0.21 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP133 (32 mg, 84% yield). 1H NMR (CDCl3) δ 8.38 (s, 1H), 8.26 (s, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.01 (d, J=8.4 Hz, 1H), 7.87 (dd, J=8.1 Hz, J=1.5 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 5.24 (s, J=6.6 Hz, 1H), 2.82 (s,

N

N NN

NH2 I

12

N

N NN

NH2

132

N

N

N

N NN

NH2 I

12

N

N NN

NH2

133

NMe

50

3H), 1.65 (d, J=6.6 Hz, 6H);ESI-MS (M+H)+ m/z calcd 319.16, found 319.10. ESI-HRMS (M+H)+ m/z calcd 319.16657, found 319.1667

Synthesis of 3-(2-chloroquinolin-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP134) A solution of 2-chloroquinolin-3-yl-3-boronic acid (72 mg, 0.35 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (40 mg, 0.14 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP134 (16 mg, 20% yield). 1H NMR (CDCl3) δ 8.37 (s, 1H), 8.26 (s, 1H), 8.10 (d, J=9.2 Hz, 1H), 7.91 (m, 1H), 7.88 (m, 1H), 7.68 (m, 1H); 5.26 (m, J=6.6 Hz, 1H), 1.66 (d, J=6.6 Hz, 6H);ESI-MS (M+H)+ m/z calcd 339.1, found 339.0. ESI-HRMS (M+H)+ m/z calcd 339.11195, found 339.112

Synthesis of 1-isopropyl-3-(1-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP135) A solution of 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (27 mg, 0.11 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP135 (11 mg, 30% yield). 1H NMR (CDCl3) δ 8.20 (s, 1H), 7.89 (m, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.46 (dd,

N

N NN

NH2 I

12

N

N NN

NH2

N

134

Cl

N

N NN

NH2 I

12

N

N NN

NH2

135

NMe

51

J=8.4 Hz, J=1.5 Hz, 1H), 7.19 (d, J=2.9 Hz, 1H), 6.58 (d, J=2.9 Hz, 1H), 5.20 (m, J=6.6 Hz, 1H), 3.88 (s, 3H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 307.2, found 307.0. ESI-HRMS (M+H)+ m/z calcd 307.16657, found 307.1667.

Synthesis of 3-(1H-indazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP137) A solution of 1H-indazol-5-yl-5-boronic acid (33 mg, 0.21 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.08 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP137 (9 mg, 21% yield). 1H NMR (CD3OD) δ 8.41 (s, 1H), 8.22 (s, 1H), 8.16 (t, J=1.5 Hz, 1H), 7.78 (m, 2H (overlapping peaks)), 5.27 (m, J=6.6 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 294.1, found 294.4. ESI-HRMS (M+H)+ m/z calcd 294.14617, found 294.1463.

Synthesis of 1-isopropyl-3-(pyrido[3,2-b]pyrazin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP139) A solution of pyrido[3,2-b]pyrazin-7-yl-7-boronic acid (ZK533, 17 mg, 0.10 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (15 mg, 0.05 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP139 (4 mg, 15% yield). 1H NMR (CDCl3) δ 9.56 (d, J=2.2 Hz, 1H), 9.16 (d, J=1.8 Hz, 1H), 9.05 (d, J=1.8 Hz, 1H), 8.72 (d,

N

N NN

NH2 I

12

N

N NN

NH2

137

NHN

N

N NN

NH2 I

12

N

N NN

NH2

N

139

N

N

52

J=2.6 Hz, 1H), 8.33 (s, 1H), 5.28 (m, J=7.0 Hz, 1H), 1.67 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 307.1, found 307.4. ESI-HRMS (M+H)+ m/z calcd 307.14142, found 307.1414.

Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-2(1H)-one (PP140) A solution of 2-hydroxyquinolin-6-yl-6-boronic acid (ZK535, 31 mg, 0.14 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP140. 1H NMR (CDCl3) δ 8.25 (s, 3H), 7.89 (d, J=9.2 Hz, 1H), 7.88 (s, 1H), 7.81 (dd, J=8.4 Hz, J=1.5 Hz, 1H), 7.58 (d, J=8.4 Hz, 1H), 6.80 (d, J=9.5 Hz, 1H), 5.22 (m, J=7.0 Hz, 1H), 1.64 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 321.1, found 321.4. ESI-HRMS (M+H)+ m/z calcd 321.14584, found 321.1467.

Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-4H-chromen-4-one (PP143) A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one (56 mg, 0.24 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.083 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in

N

N NN

NH2 I

12

N

N NN

NH2

140

HNO

N

N NN

NH2 I

12

N

N NN

NH2

143

O

O

53

vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP143 (12 mg, 26% yield). 1H NMR (CD3OD) δ 8.46 (d, J=2.2 Hz, 1H), 8.40 (s, 1H), 8.27 (d, J=6.2 Hz, 1H), 8.16 (dd, J=8.8 Hz, J=2.2 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 6.47 (d, J=6.2 Hz, 1H), 5.26 (m, J=6.6 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 322.1, found 322.4. ESI-HRMS (M+H)+ m/z calcd 322.12985, found 322.1299

Synthesis of tert-butyl 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-4-ylcarbamate (PP145) A solution of tert-butyl quinolin-4-ylcarbamate boronic acid (95 mg, 0.33 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.17 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield BA145 (20 mg, 18% yield). ESI-MS (M+H)+ m/z calcd 420.2, found 420.5. ESI-HRMS (M+H)+ m/z calcd 420.21425, found 420.2144. Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-4-amine (PP147) tert-butyl 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-4-ylcarbamate (PP145, 15 mg, 0.04 mmol) was dissolved in 50% TFA in CH2Cl2 and stirred at room temperature for 90 minutes. The reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield BA147 (3 mg, 14% yield). 1H NMR (CD3OD) δ 8.71 (d, J=1.8 Hz, 1H), 8.39 (s, 1H), 8.38 (d, J=6.6 Hz, 1H), 8.29 (dd, J=8.8 Hz, J=1.8 Hz, 1H), 8.03 (d, J=8.8 Hz, 1H), 6.92 (d, J=7.0 Hz, 1H), 5.26 (m, J=6.6 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 320.2, found 320.4. ESI-HRMS (M+H)+ m/z calcd 320.16182, found 320.1619.

N

N NN

NH2 I

12

N

N NN

NH2

147

N

NH O

O

N

N NN

NH2

N

NH2

145

54

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2,6-difluorobenzaldehyde (PP149) A solution of 3,5-difluoro-4-formyl-phenylboronic acid (150 mg, 0.83 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP149 (33 mg, 31% yield). ESI-MS (M+H)+ m/z calcd 318.1, found 318.4. ESI-HRMS (M+H)+ m/z calcd 318.11609, found 318.1171.

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (PP150) A solution of 4-cyano-3-fluorophenylboronic acid (136 mg, 0.83 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP150 (50 mg, 51% yield). ESI-MS (M+H)+ m/z calcd 297.1, found 297.4. ESI-HRMS (M+H)+ m/z calcd 297.12585, found 297.1259.

N

N NN

NH2 I

12

N

N NN

NH2

149

CHO

FF

N

N NN

NH2 I

12

N

N NN

NH2

150

FNC

55

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (PP151) A solution of 4-cyano-3-fluorophenylboronic acid (135 mg, 0.83 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP151 (47 mg, 48 % yield). ESI-MS (M+H)+ m/z calcd 297.1, found 297.4. ESI-HRMS (M+H)+ m/z calcd 297.12585, found 297.1267.

Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzaldehyde (PP152) A solution of 3-fluoro-4-formylphenyl boronic acid (140 mg, 0.83 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP152 (25 mg, 25 % yield). 1H NMR (DMSO-d6) δ 10.30 (s, 1H), 8.27 (s, 1H), 7.98 (t, J=7.7 Hz, 1H), 7.69 (dd, J=8.1 Hz, J=1.1 Hz, 1H), 7.62 (dd, J=11.4 Hz, J=1.5 Hz, 1H), 5.09 (m, J=6.6 Hz, 1H), 1.50 (d, J=6.6 Hz, 1H), 1.50 (J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 300.1, found 300.4. ESI-HRMS (M+H)+ m/z calcd 300.12552, found 300.1256.

N

N NN

NH2 I

12

N

N NN

NH2

151

F CN

N

N NN

NH2 I

12

N

N NN

NH2

152

F CHO

56

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-benzo[d]imidazol-2(3H)-one (PP156) A solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2(3H)-one (54 mg, 0.21 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.33 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP156 (6 mg, 14 % yield). 1H NMR (CD3OD) δ 8.36 (s, 1H), 7.40 (m, 1H), 7.39 (m, 1H), 7.26 (d, J=8.4 Hz, 1H), 5.23 (m, J=6.6 Hz, 1H), 1.62 (d, J=6.6 Hz, 6H). ESI-HRMS (M+H)+ m/z calcd 310.14108, found 310.1412.

Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinazolin-4(3H)-one (PP158)

A solution of 3,4-dihydro-4-oxoquinazolin-6-yl-6-boronic acid (ZK532, 30 mg, 0.16 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.08 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP158 (8 mg, 20 % yield). 1H NMR (CD3OD) δ 8.57 (d, J=1.8 Hz, 1H), 8.41 (s, 1H), 8.23 (s, 1H), 8.22 (dd, J=8.4 Hz, J=2.2 Hz, 1H), 7.92 (d, J=8.4 Hz, 1H), 5.27 (m, J=6.6 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 322.1, found 322.4. ESI-HRMS (M+H)+ m/z calcd 322.14108, found 322.142.

N

N NN

NH2 I

12

N

N NN

NH2

156

NHHN O

N

N NN

NH2 I

12

N

N NN

NH2

158

NHN

O

57

Synthesis of 3-(H-imidazo[1,2-a]pyridin-7-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP161) A solution of imidazo[1,2-a]pyridine-6-boronic acid (33 mg, 0.21 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.08 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP161 (26 mg, 81 % yield). 1H NMR (CD3OD) δ 9.22 (t, J=1.1 Hz, 1H), 8.45 (s, 1H), 8.37 (d, J=1.5, 1H), 8.30 (dd, J=9.2 Hz, J=1.5 Hz, 1H), 8.18 (d, J=2.2 Hz, 1H), 8.15 (d, J=9.5 Hz, 1H), 5.30 (m, J=6.6 Hz, 1H), 1.64 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 294.1, found 294.4. ESI-HRMS (M+H)+ m/z calcd 294.14617, found 294.1462.

Synthesis of 1-cyclopentyl-3-(H-imidazo[1,2-a]pyridin-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP162) A solution of imidazo[1,2-a]pyridine-6-boronic acid (25 mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA80, 20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP162. 1H NMR (CD3OD) δ 8.45 (s, 1H), 8.37 (d, J=2.2 Hz, 1H), 8.29 (dd, J=9.5 Hz, J=1.5 Hz, 1H), 8.18 (d, J=2.2 Hz, 1H), 8.15

N

N NN

NH2 I

12

N

N NN

NH2

N

161

N

N

N NN

NH2 I

80

N

N NN

NH2

N

162

N

58

(dt, J=1.5 Hz, J=1.1 Hz, 1H), 5.46 (m, J=7.3 Hz, 1H), 2.23 (m, 4H), 2.04 (m, 2H), 1.84 (m, 2H); ESI-MS (M+H)+ m/z calcd 320.2, found 320.4. ESI-HRMS (M+H)+ m/z calcd 320.16182, found 320.162.

Synthesis of 1-cyclopentyl-3-(quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP112) A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (58 mg, 0.23 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.09 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP112 (42 mg, 84% yield). 1H NMR (CDCl3) δ 9.22 (d, J=3.7 Hz, 1H), 8.75 (d, J=8.1 Hz, 1H), 8.59 (d, J=8.8 Hz, 1H), 8.32 (d, J=1.8 Hz, 1H), 8.26 (dd, J=8.8 Hz, J=1.5 Hz, 1H), 7.88 (dd, J=8.4 Hz, J=4.8 Hz, 1H). 5.40 (m, J=7.3 Hz, 1H), 2.23 (m, 4H), 2.04 (m, 2H), 1.80 (m, 2H); ESI-MS (M+H)+ m/z calcd 331.2, found 331.1. ESI-HRMS (M+H)+ m/z calcd 331.16657, found 331.1667.

Synthesis of 1-cyclopentyl-3-(1H-indazol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP115) A solution of 6-indazolboronic acid (25 mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP115 (18 mg, 56% yield). 1H NMR (CDCl3) δ 8.28 (s,

N

N NN

NH2 I

80

N

N NN

NH2

112

N

N

N NN

NH2 I

80

N

N NN

NH2

115

NHN

59

1H), 8.21 (s, 1H), 8.49 (d, J=8.1 Hz, 1H), 7.85 (s, 1H), 7.45 (dd, J=8.1 Hz, J=0.7 Hz, 1H), 5.36 (m, J=7.4 Hz, 1H), 2.23 (m, 4H), 2.03 (m, 2H), 1.78 (m, 2H); ESI-MS (M+H)+ m/z calcd 320.2, found 320.0. ESI-HRMS (M+H)+ m/z calcd 320.16182, found 320.162.

Synthesis of 1-cyclopentyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP121) A solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (44 mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP121 (27 mg, 80% yield). 1H NMR (DMSO-d6) δ 11.83 (br, 1H), 8.48 (d, J=2.1 Hz, 1H), 8.24 (s, 1H), 8.19 (d, J=1.79 Hz, 1H), 7.57 (d, J=3.2 Hz, 1H), 6.57 (d, J=3.2 Hz, 1H), 5.25 (m, J=7.4 Hz, 1H), 2.09 (m, 4H), 1.91 (m, 2H), 1.70 (m, 2H); 13C NMR (DMSO-d6) δ 158.9, 156.1, 154.5, 149.0, 143.1, 142.8, 128.5, 127.8, 121.7, 120.2, 101.1, 98.4, 57.5, 32.5, 24.9; ESI-MS (M+H)+ m/z calcd 320.2, found 320.0. ESI-HRMS (M+H)+ m/z calcd 320.1624, found 320.1609.

Synthesis of 1-cyclopentyl-3-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP122) A solution of quinolin-3-yl-3-boronic acid (26 mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC

N

N NN

NH2 I

80

N

N NN

NH2

N

121

HN

N

N NN

NH2 I

80

N

N NN

NH2

N

122

60

(MeCN:H2O:0.1% TFA) to yield PP122 (26 mg, 78% yield). 1H NMR (CDCl3) δ 9.65 (d, J=2.2 Hz, 1H), 9.08 (d, J=1.5 Hz, 1H), 8.38 (d, J=8.8 Hz, 1H), 8.23 (s, 1H), 8.19 (d, J=7.7 Hz, 1H), 8.05 (m, 1H), 7.90 (m, 1H), 5.41 (m, J=7.3 Hz, 1H), 2.24 (m, 4H), 2.04 (m, 2H), 1.81 (m, 2H). ESI-HRMS (M+H)+ m/z calcd 331.16657, found 331.1667.

Synthesis of 1-cyclopentyl-3-(1H-indol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP124) A solution of indole-6-boronic acid (25 mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP124 (20 mg, 62% yield). 1H NMR (CD3OD) δ 8.36 (m, 1H), 7.76 (d, J=7.9 Hz, 1H), 7.73 (s, 1H), 7.39 (m, 1H), 7.33 (d, J=8.3 Hz, 1H), 5.38 (m, J=7.4 Hz, 1H), 2.20 (m, 4H), 2.02 (m, 2H), 1.79 (m, 2H). ESI-HRMS (M+H)+ m/z calcd 319.16657, found 319.1667.

Synthesis of 1-cyclopentyl-3-(1-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP136) A solution of 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (49 mg, 0.19 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.075 mmol) in DME (6 mL). Pd(PPh3)4 (8

N

N NN

NH2 I

80

N

N NN

NH2

124

NH

N

N NN

NH2 I

80

N

N NN

NH2

136

NMe

61

mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP136 (16 mg, 38% yield). 1H NMR (CDCl3) δ 8.20 (s, 1H), 7.88 (m, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.45 (dd, J=8.1 Hz, J=1.8 Hz, 1H), 7.19 (d, J=2.9 Hz, 1H), 6.58 (dd, J=2.9 Hz, J=0.7 Hz, 1H), 5.32 (m, J=7.7 Hz, 1H), 3.88 (s, 3H), 2.22 (m, 4H), 2.03 (m, 2H), 1.78 (m, 2H); ESI-MS (M+H)+ m/z calcd 333.2, found 333.0. ESI-HRMS (M+H)+ m/z calcd 333.18222, found 333.1823.

Synthesis of 1-cyclopentyl-3-(1H-indazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP138) A solution of 1H-indazol-5-yl-5-boronic acid (25mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP138 (16 mg, 54% yield). 1H NMR (CD3OD δ 8.41 (s, 1H), 8.21 (s, 1H), 8.14 (t, J=1.5 Hz, 1H), 7.77 (m, 2H (overlapping peaks)), 5.42 (m, J=7.7 Hz, 1H), 2.23 (m, 4H), 2.05 (m, 2H), 1.82 (m, 2H); ESI-MS (M+H)+ m/z calcd 320.2, found 320.5. ESI-HRMS (M+H)+ m/z calcd 320.16182, found 320.1619.

Synthesis of 6-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-2(1H)-one (PP141)

N

N NN

NH2 I

80

N

N NN

NH2

138

NHN

N

N NN

NH2 I

80

N

N NN

NH2

141

HNO

62

A solution of 2-hydroxyquinolin-6-yl-6-boronic acid (ZK353, 25mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP141. 1H NMR (CDCl3) δ 8.26 (s, 1H), 7.87 (d, J=6.2 Hz, 1H), 7.85 (d, J=1.5 Hz, 1H), 7.79 (dd, J=8.4 Hz, J=1.8 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 6.80 (d, J=9.5 Hz, 1H), 5.34 (m, J=7.7 Hz, 1H), 2.21 (m, 4H), 2.02 (m, 2H), 1.79 (m, 2H); ESI-MS (M+H)+ m/z calcd 347.2, found 347.5. ESI-HRMS (M+H)+ m/z calcd 347.16149, found 347.1616.

Synthesis of 6-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-4H-chromen-4-one (PP144) A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one (25mg, 0.15 mmol) in EtOH (1.65 mL) was added to a solution of 1-cyclopentyl-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20 mg, 0.06 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP144 (25 mg, 89% yield). 1H NMR (CDCl3) δ 8.48 (d, J=2.2 Hz, 1H), 8.24 (s, 1H), 8.00 (dd, J=8.8 Hz, J=2.2 Hz, 1H), 7.91 (d, J=6.2 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 6.36 (d, J=6.2 Hz, 1H), 5.35 (m, J7.7 Hz, 1H), 2.20 (m, 4H), 2.02 (m, 2H), 1.78 (m, 2H); ESI-MS (M+H)+ m/z calcd 348.1, found 348.4. ESI-HRMS (M+H)+ m/z calcd 348.1455, found 348.1455

N

N NN

NH2 I

80

N

N NN

NH2

144

O

O

N

N NN

NH2 I

Me

N

N NN

NH2

Me109 111

N

63

Synthesis of 1-methyl-3-(quinolin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP111) A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (46mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (BA109, 20mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP111 (13 mg, 37% yield). 1H NMR (CDCl3) δ 9.16 (dd, J=4.8 Hz, J=1.5 Hz, 1H), 8.51 (m, 1H), 8.49 (m, 1H), 8.31 (s, 1H), 8.21 (d, J=2.2 Hz, 1H), 8.13 (dd, J=8.8 Hz, 1.8 Hz, 1H), 7.74 (dd, J=8.4 Hz, 4.4 Hz, 1H), 4.20 (s, 3H); ESI-MS (M+H)+ m/z calcd 277.1, found 277.1. ESI-HRMS (M+H)+ m/z calcd 277.11962, found 277.1198.

Synthesis of 3-(2-ethoxynaphthalen-6-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP116) A solution of 6-ethoxy-2-naphthaleneboronic acid (39mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP116 (19 mg, 63% yield). 1H NMR (CDCl3) δ 8.26 (s, 1H), 8.02 (s, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.81 (d, J=8.8 Hz, 1H), 7.66 (dd, J=8.4 Hz, 1.8 Hz, 1H), 7.27 (dd, J=8.1 Hz, J=2.6 Hz, 1H), 7.20 (s, 1H), 4.20 (q, J=7.0 Hz, 2H), 4.16 (s, 3H), 1.52 (t, J=7.0 Hz, 3H); ESI-MS (M+H)+ m/z calcd 320.1, found 320.1. ESI-HRMS (M+H)+ m/z calcd 320.15059, found 320.1507.

N

N NN

NH2 I

Me

N

N NN

NH2

Me109 116

OEt

64

Synthesis of 3-(1H-indol-6-yl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP119)

A solution of indole-6-boronic acid (29mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP119 (25 mg, 71% yield). 1H NMR (CDCl3) δ 8.25 (s, 1H), 7.83 (d, J=8.1 Hz, 1H), 7.69 (d, J=0.7 Hz, 1H), 7.38 (m, 1H), 7.34 (dd, J=8.1 Hz, J=1.5 Hz, 1H), 6.67 (m, 1H), 4.14 (s, 3H); ESI-MS (M+H)+ m/z calcd 265.1, found 265.1. ESI-HRMS (M+H)+ m/z calcd 265.11962, found 265.1197.

Synthesis of 1-methyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP120)

A solution of 7-azaindole-5-boronic acid (53 mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP120 (11 mg, 31% yield). 1H NMR (CDCl3) δ 8.58 (d, J=1.8 Hz, 1H), 8.42 (d, J=1.8 Hz, 1H), 8.31 (s, 1H), 7.61 (dd, J=3.3 Hz, J=2.2 Hz, 1H), 6.77 (dd, J=3.3 Hz, J=1.8 Hz, 1H), 4.18 (s, 3H); ESI-MS (M+H)+ m/z calcd 266.1, found 266.0. ESI-HRMS (M+H)+ m/z calcd 266.11487, found 266.1149.

N

N NN

NH2 I

Me

N

N NN

NH2

Me109 119

NH

N

N NN

NH2 I

Me

N

N NN

NH2

N

Me109 120

HN

65

N

N NN

NH2 I

109

N

N NN

NH2

148

O

O

Synthesis of 6-(4-amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-4H-chromen-4-one (PP148)

A solution of A solution of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one (50 mg, 0.18 mmol) in EtOH (1.65 mL) was added to a solution of 3-iodo-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (20mg, 0.07 mmol) in DME (6 mL). Pd(PPh3)4 (8 mg, 0.007 mmol) and saturated Na2CO3 (0.95 mL) were added and the reaction was heated to 90°C under an argon atmosphere overnight. After cooling, the reaction was extracted with saturated NaCl and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP148 (6 mg, 28% yield). 1H NMR (DMSO-d6) δ 8.36 (d, J=6.2 Hz, 1H), 8.29 (d, 1H), 8.25 (d, J=3.3 Hz, 1H), 8.06 (dd, J=8.8 Hz, J=2.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 6.42 (d, J=5.9 Hz, 1H), 3.98 (s, 3H); ESI-MS (M+H)+ m/z calcd 294.1, found 294.4. ESI-HRMS (M+H)+ m/z calcd 294.09855, found 294.0986.

N

N NN

NH2

130

NCl

N

N NN

NH2

HNN

142

NH2

Synthesis of 3-(2-(hydrazine)quinolin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP142)

3-(2-chloroquinolin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP130, 20 mg, 0.05 mmol) was dissolved in 1 mL EtOH under an argon atmosphere. Hydrazine (1.6 mL, 33 mmol) was added by syringe and reaction was heated to 90°C for 6.5 hours. Crude reaction was cooled, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP142 (12 mg, 61% yield). 1H NMR (CDCl3) δ 8.27 (s, 1H), 8.20 (d, J=8.8 Hz, 1H), 8.02-7.97 (m, 3H (overlapping spectra)), 7.84 (d, J=10.6 Hz, 1H), 5.24 (m, J=7.0 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 335.2, found 335.5. ESI-HRMS (M+H)+ m/z calcd 335.17272, found 335.1728.

66

Synthesis of 6-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)quinolin-2-amine (PP146)

3-(2-chloroquinolin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP130, 50 mg, 0.15 mmol), acetamide (174 mg, 3.0 mmol) and K2CO3 (104 mg, 0.75 mmol) were combined and heated to 200°C under an argon atmosphere for one hour. Reaction was cooled, then extracted with H2O and CH2Cl2. Organic phases were combined, concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP146 (22 mg, 46 % yield). 1H NMR (CD3OD) δ 8.48 (d, J=9.2 Hz, 1H), 8.37 (s, 1H), 8.25 (d, J=1.8 Hz, 1H), 8.16 (dd, J=8.8 Hz, J=1.8 Hz, 1H), 7.86 (d, J=8.4 Hz, 1H), 5.24 (m, J=6.6 Hz, 1H), 1.63 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 320.2, found 320.4. ESI-HRMS (M+H)+ m/z calcd 320.16182, found 320.1619.

Synthesis of 3-(3-amino-1H-indazol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP154)

4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (PP150, 20 mg, 0.07 mmol) was dissolved in n-BuOH (2 mL). Hydrazine monohydrate (0.400 mL) was added and the reaction was heated to 110°C under an argon atomosphere and left stirring over night. Reaction mixture was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP154 (15 mg, 70 % yield). 1H NMR (CD3OD) δ 8.43 (s, 1H), 8.07 (dd, J=8.4 Hz, J=0.7 Hz, 1H), 7.75 (m, 1H), 7.52 (dd, J=8.4 Hz, J=1.5 Hz, 1H), 5.28 (m, J=6.6 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 309.2, found 309.4. ESI-HRMS (M+H)+ m/z calcd 309.15707, found 309.1572. Synthesis of 4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-hydroxybenzonitrile (PP155)

N

N NN

NH2

130

NCl

N

N NN

NH2

NNH2

146

N

N NN

NH2

154150

N

N NN

NH2

NH

N

N NN

NH2

OH

155

F

NNN

H2N

67

4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (BA150, 25 mg, 0.1 mmol) was dissolved in DMF (1 mL). t-BuOK (24 mg, 0.21 mmol) was added and the reaction was stirred at room temperature overnight. Reaction was then heated to 150°C for 24 hours. The reaction was then concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP155 (21 mg, 89 % yield). 1H NMR (CD3OD) δ 8.41 (s, 1H), 7.75 (m, 1H), 7.32 (d, J=1.5 Hz, 1H), 7.30 (dd, J=8.2 Hz, J=1.5 Hz, 1H), 5.26 (m, J=7.0 Hz, 1H), 1.62 (d, J=7.0 Hz, 6H); ESI-MS (M+H)+ m/z calcd 295.1, found 295.4. ESI-HRMS (M+H)+ m/z calcd 295.13019, found 295.1302.

Synthesis of 3-(3-aminobenzo[d]isoxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP257) & 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-hydroxybenzonitrile (PP157) 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (BA151, 20 mg, 0.07 mmol) was dissolved in DMF (1 mL). t-BuOK (24 mg, 0.21 mmol) was added and the reaction was stirred at room temperature overnight. Reaction was then heated to 150°C for 24 hours. The reaction was then concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP257 (7 mg), 1H NMR (CD3OD) δ 8.36 (s, 1H), 7.88 (d, J=2.2 Hz, 1H), 7.83 (dd, J=8.4 Hz, J=2.2 Hz, 1H), 7.18 (d, J=8.1 Hz, 1H), 5.22 (m, J=7.0 Hz, 1H), 1.62 (d, J=6.6 Hz, 6H), ESI-MS (M+H)+ m/z calcd 295.1, found 295.4; ESI-HRMS (M+H)+ m/z calcd 295.13019, found 295.1312 and PP157 (8 mg), 1H NMR (CDCl3) δ 8.25 (s, 1H), 7.87 (s, 1H), 7.80 (dd, J=8.4 Hz, J=1.5 Hz, 1H), 7.62 (d, J=8.4 Hz, 1H), 5.23 (m, J=7.0 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 310.1, found 310.4. ESI-HRMS (M+H)+ m/z calcd 310.14108, found 310.1422. Synthesis of 3-(3-amino-1H-indazol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP159)

4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2,6-difluorobenzaldehyde (PP149, 20 mg, 0.063 mmol) was dissolved in n-BuOH (1 mL).

N

N NN

NH2

257151

N

N NN

NH2

F NO

NH2

N

N NN

NH2

NOH

N

157

N

N NN

NH2

CHO

FF

N

N NN

NH2

FNHN

149 159

68

Hydrazine monohydrate (0.400 mL) was added and the reaction was heated to 100°C under an argon atomosphere and left stirring for 2.5 hours. Reaction mixture was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP159 (15 mg, 77 % yield). 1H NMR (CD3OD) δ 8.41 (s, 1H), 8.26 (d, J=1.1 Hz, 1H), 7.72 (s, 1H), 7.22 (dd, J=10.3 Hz, J=1.1 Hz, 1H), 5.27 (m, J=7.0 Hz, 1H), 1.64 (d, J=6.6 Hz, 6H); ESI-MS (M+H)+ m/z calcd 312.1, found 312.4. ESI-HRMS (M+H)+ m/z calcd 312.13675, found 312.1369.

N

N NN

NH2

F

CN

N

N NN

NH2

NHN

151 160

NH2

Synthesis of 3-(3-amino-1H-indazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP160)

4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorobenzonitrile (PP151, 17 mg, 0.06 mmol) was dissolved in n-BuOH (2 mL). Hydrazine monohydrate (0.500 mL) was added and the reaction was heated to 110°C under an argon atomosphere overnight. Reaction mixture was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP160 (18 mg, 100% yield), 1H NMR (CD3OD) δ 8.42 (s, 1H), 8.23 (m, 1H), 7.88 (dd, J=8.8 Hz, 1.5 Hz, 1H), 7.62 (dd, J=8.8 Hz, J=0.7 Hz, 1H), 5.28 (m, J=7.0 Hz, 1H), 1.64 (d, J=7.0 Hz, 6H), ESI-MS (M+H)+ m/z calcd 309.2, found 309.4. ESI-HRMS (M+H)+ m/z calcd 309.15707, found 309.1573.

N

N N

ICl

Me

N

N N

Cl

Me

223

69

Synthesis of 4-chloro-7-methyl-5-(naphthalen-2-yl)-7H-pyrrolo[2,3-d]pyrimidine (PP223) A solution of 4-chloro-5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (19 mg, 0.065 mmol), naphthalen-2-yl-2-boronic acid (12.2 mg, 0.071 mmol), Na2CO3 (68.9 mg, 0.65 mmol) and PdCl2(dppf) (26.5 mg, 0.00325 mmol) in THF (3 mL) was heated to reflux overnight under an argon atmosphere. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP223 (5 mg, 26% yield). ESI-MS (M+H)+ m/z calcd 294.1, found 294.3.

Synthesis of 4-chloro-7-methyl-5-(3-biphenyl)-7H-pyrrolo[2,3-d]pyrimidine (ZK103) A solution of 4-chloro-5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (10 mg, 0.034 mmol), 3-biphenyl-boronic acid (7.4 mg, 0.038 mmol), Na2CO3 (36.1 mg, 0.34 mmol) and PdCl2(dppf) (1.4 mg, 0.0017 mmol) in THF (10 mL) was heated to reflux overnight under an argon atmosphere. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield ZK103 (3 mg, 28% yield). ESI-MS (M+H)+ m/z calcd 320.1, found 320.0.

Synthesis of 3-(4-tert-butylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP125) 1-tert-butyl-3-(4-tert-butylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux for 2 hours. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield PP125 (quant.). 1H NMR (4:1 CDCl3:CD3OD) δ 8.31 (s, 1H), 7.62–7.57 (m, 4H), 1.39 (s, 1H). ESI-MS (M+H)+ m/z calcd 268.2, found 268.4.

N

N NN

NH2

N

N NH

N

NH2

125

N

N N

ICl

Me

N

N N

Cl

Me

103

70

Synthesis of 3-(3-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP126) 1-tert-butyl-3-(3-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.16 mmol) was dissolved in a solution of formic acid (5 mL) and conc. HCl (0.1 mL) and heated to reflux for 2.5 hours. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.32 (s, 1H), 7.59–7.54 (m, 1H), 7.44–7.36 (m, 3H), 7.26–7.24 (m, 1H), 7.20–7.15 (m, 2H), 7.11–7.08 (m, 2H). ESI-MS (M+H)+ m/z calcd 304.1, found 304.3.

Synthesis of 3-m-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP204) 1-tert-butyl-3-m-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (23 mg, 0.1 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.3 mL) and heated to reflux for 2.5 hours. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.32 (s, 1H), 7.50–7.44 (m, 3H), 7.39–7.37 (m, 1H), 2.47 (s, 3H). ESI-MS (M+H)+ m/z calcd 226.1, found 226.3.

Synthesis of 3-(3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP128) 1-tert-butyl-3-(3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (23 mg, 0.055 mmol) was dissolved in a solution of formic acid (5 mL) and conc. HCl (0.1 mL) and heated to reflux for 2 hours. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.58 (t, J=2.0 Hz, 1H), 8.44–8.40 (m, 1H), 8.38 (s, 1H), 8.11–8.08 (m, 1H), 7.83 (t, J=8.0 Hz, 1H). ESI-MS (M+H)+ m/z calcd 257.1, found 257.3.

N

N NN

NH2

N

N NH

N

NH2

126

O O

N

N NN

NH2

N

N NH

N

NH2

204

Me Me

N

N NN

NH2

N

N NH

N

NH2

128

NO2 NO2

71

Synthesis of 3-(benzo[d][1,3]dioxol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP233) 1-tert-butyl-3-(benzo[d][1,3]dioxol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (21 mg, 0.082 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.2 mL) and heated to reflux for 2 hours. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (3:1 CDCl3:CD3OD) δ 8.32 (s, 1H), 7.14–7.11 (m, 2H), 7.02–7.00 (m, 1H), 6.10 (s, 2H). ESI-MS (M+H)+ m/z calcd 256.1, found 256.3.

Synthesis of 3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP213) 1-tert-butyl-3-(4-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (21 mg, 0.082 mmol) was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 257.1, found 257.3.

Synthesis of 3-(3-(2,6-dichlorobenzyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP218) 1-tert-butyl-3-(3-(2,6-dichlorobenzyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (19.5 mg, 0.05 mmol) was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and

N

N NN

NH2

N

N NH

N

NH2

218

O OCl

Cl

Cl

Cl

N

N NN

NH2

OO

N

N NH

N

NH2

OO

129

N

N NN

NH2

N

N NH

N

NH2

213

NO2 NO2

72

purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.33 (s, 1H), 7.55 (t, J=8.0 Hz, 1H), 7.43 (d, J=1.0 Hz, 1H), 7.41 (s, 1H), 7.36–7.29 (m, 3H), 7.25–7.21 (m, 1H), 5.39 (s, 2H). ESI-MS (M+H)+ m/z calcd 386.1, found 386.2.

Synthesis of 3-(2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP206) 1-tert-butyl-3-(2,3-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (34 mg, 0.14 mmol) was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.34 (s, 1H), 7.39–7.36 (m, 1H), 7.29 (t, J=7.5 Hz, 1H), 7.23–7.20 (m, 1H), 2.40 (s, 3H), 2.18 (s, 3H). ESI-MS (M+H)+ m/z calcd 240.1, found 240.4.

Synthesis of 2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (PP210) 1-tert-butyl-2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (5 mg, 0.014 mmol) was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 228.1, found 228.3.

N

N NN

NH2

N

N NH

N

NH2

205

Me Me

Synthesis of 3-o-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP205) 1-tert-butyl-3-o-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.34 (s, 1H), 7.50–7.41 (m, 2H), 7.40–7.37 (m, 2H), 2.31 (s, 3H). ESI-MS (M+H)+ m/z calcd 226.1, found 226.3.

N

N NN

NH2

N

N NH

N

NH2

206

Me Me

MeMe

N

N NN

NH2

N

N NH

N

NH2

210

OHOBn

73

Synthesis of 3-(3-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP212) 1-tert-butyl-3-(3-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in a solution of formic acid (2 mL) and conc. HCl (0.2 mL) and heated to reflux for 30 min. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 227.1, found 227.3. Synthesis of 3-(3-(benzyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP217) 1-tert-butyl-3-(3-(benzyloxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (19 mg, 0.052 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux for 30 min. Reaction yielded a mixture of PP217 and 3-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenol (PP207). Reaction was concentrated in vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). PP217: ESI-MS (M+H)+ m/z calcd 318.1, found 318.3. PP207: 1H NMR (1:1 CDCl3:CD3OD) δ 8.35 (s, 1H), 7.43 (t, J=7.8 Hz, 1H), 7.14–7.10 (m, 2H), 7.05–7.02 (m, 1H). ESI-MS (M+H)+ m/z calcd 228.1, found 228.3.

Synthesis of 3-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP211) 1-tert-butyl-3-(4-aminophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9 mg, 0.032 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.2 mL) and heated to reflux. The reaction was allowed to proceed 30 min., then concentrated in

N

N NN

NH2

N

N NH

N

NH2

211

NH2 NH2

N

N NN

NH2

N

N NH

N

NH2

212

NH2 NH2

N

N NN

NH2

N

N NH

N

NH2

217

OBn OBn

N

N NN

NH2

OH

207

74

vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 227.1, found 227.3.

Synthesis of 3-(1,2,3,4-tetrahydronaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP221) 1-tert-butyl-3-(1,2,3,4-tetrahydronaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (9 mg, 0.029 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux. The reaction was allowed to proceed 30 min., then concentrated in vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (1:1 CDCl3:CD3OD) δ 8.25 (s, 1H), 7.16–7.10 (m, 4H), 3.61–3.51 (m, 1H), 3.17 (d, J=8.0 Hz, 2H), 3.13–2.92 (m, 2H), 2.36–2.28 (m, 1H), 2.16–2.02 (m, 1H). ESI-MS (M+H)+ m/z calcd 266.1, found 266.4.

Synthesis of 5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (ZK140) 4-chloro-5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (90 mg, 0.31 mmol) was taken up in 7N NH3/MeOH and heated in a sealed tube at 110º C overnight. The reaction was concentrated in vacuo to give a brown/off-white solid.

Synthesis of 3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP201) 1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux. The reaction

N

N NN

Me

NH2

N

N NH

N

Me

NH2

201

N

N NN

NH2

N

N NH

N

NH2

221

N

N N

ICl

Me

N

N N

INH2

Me140

75

was allowed to proceed 30 min., then concentrated in vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 226.1, found 226.3.

Synthesis of 3-(4-biphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP220) 1-tert-butyl-3-(4-biphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (22 mg, 0.066 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux. The reaction was allowed to proceed 30 min., then concentrated in vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (DMSO–d6) δ 8.39 (br, 1H), 7.88 (d, J=8.1 Hz, 2H), 7.80–7.75 (m, 4H), 7.54–7.50 (m, 2H), 7.44–7.40 (m, 1H). ESI-MS (M+H)+ m/z calcd 288.1, found 288.3.

Synthesis of 3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP215) 1-tert-butyl-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (19 mg, 54.1 mmol) was dissolved in a solution of formic acid (1 mL) and conc. HCl (0.1 mL) and heated to reflux. The reaction was allowed to proceed 30 min., then concentrated in vacuo and the products purified by RP-HPLC (MeCN:H2O:0.1% TFA). 1H NMR (DMSO–d6) δ 8.38 (br, 1H), 7.70–7.66 (m, 2H), 7.49–7.42 (m, 2H), 7.24–7.12 (m, 5H). ESI-MS (M+H)+ m/z calcd 304.1, found 304.3.

N

N NN

NH2

N

N NH

N

NH2

215

O O

N

N NN

NH2

N

N NH

N

NH2

220

76

Synthesis of 1-benzyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP229) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (110 mg, 0.42 mmol) was dissolved in DMF (2 mL) and K2CO3 (220 mg, 1.6 mmol) and benzyl bromide (71.8 mg, 0.42 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration and then purified further by silica gel chromatography (5% MeOH/CH2Cl2) to yield a white solid.

N

N N

Cl

Me

N

N N

NH2

Me

N

N N

ICl

Me

O O

216 Synthesis of 5-(4-(benzyloxy)phenyl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (PP216) A solution of 5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (5 mg, 0.018 mmol), 4-(benzyloxy)phenylboronic acid (21 mg, 0.091 mmol), K3PO4 (19.3 mg, 0.091 mmol) and Pd(PPh3)4 (12.5 mg, 0.011 mmol) in DMF (3 mL) was heated to 60º C under an argon atmosphere. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 331.1, found 331.3.

Synthesis of 5-(3-biphenyl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (PP219) A solution of 5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (5 mg, 0.018 mmol), 3-biphenylboronic acid (18 mg, 0.091 mmol), K3PO4 (19.3 mg, 0.091 mmol) and Pd(PPh3)4 (12.5 mg, 0.011 mmol) in DMF (3 mL) was heated to 60º C under an argon atmosphere. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 301.1, found 301.3.

N

N NH

N

NH2

229

N

N NN

NH2

N

N N

INH2

Me

N

N N

NH2

Me

219

77

Synthesis of 5-(benzo[b]thiophen-2-yl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (PP234) A solution of 5-iodo-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (5 mg, 0.018 mmol), benzo[b]thiophen-2-yl-2-boronic acid (16 mg, 0.091 mmol), K3PO4 (19.3 mg, 0.091 mmol) and Pd(PPh3)4 (12.5 mg, 0.011 mmol) in DMF (3 mL) was heated to 60º C under an argon atmosphere. Reaction was concentrated in vacuo and purified by RP-HPLC (MeCN:H2O:0.1% TFA). ESI-MS (M+H)+ m/z calcd 281.1, found 281.3.

Synthesis of 3-(naphthalen-2-yl)-1-phenethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP230) 2-(methoxy(naphthalen-6-yl)methylene)malononitrile (100 mg, 0.43 mmol) and phenethylhydrazine hydrogen chloride (58.5 mg, 0.43 mmol) were dissolved in EtOH (3 mL) and TEA (60 μL, 0.43 mmol) and heat to reflux for one hour. The product was extracted with diethylether and concentrated in vacuo. This concentrate was then dissolved in formamide (10 mL) and heated to 160-180º C overnight. The following day the reaction was cooled, poured into water, and the precipitated product collected by filtration. ESI-MS (M+H)+ m/z calcd 366.2, found 366.2.

N

N N

INH2

Me

N

N N

NH2

Me

234

S

N

N NH

N

NH2

N

N NN

NH2

226

N

N NH

N

NH2

N

N NN

NH2

230

78

Synthesis of 1-isopropyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP226) 2-(methoxy(naphthalen-6-yl)methylene)malononitrile (100 mg, 0.43 mmol) and isopropylhydrazine hydrogen chloride (47.3 mg, 0.43 mmol) were dissolved in EtOH (3 mL) and TEA (1 eq.) and heat to reflux for one hour. The product was extracted with diethylether and concentrated in vacuo. This concentrate was then dissolved in formamide (10 mL) and heated to 160-180º C overnight. The following day the reaction was cooled, poured into water, and the precipitated product collected by filtration. 1H NMR (DMSO–d6) δ 8.40 (s, 1H), 8.21 (m, 1H), 8.10 (d, J=8.6 Hz, 1H), 8.07–8.01 (m, 2H), 7.82 (dd, J=8.6, 1.8 Hz, 1H), 7.63–7.58 (m, 2H), 5.21–5.06 (m, 1H), 1.54 (d, J=6.7 Hz, 6H). ESI-MS (M+H)+ m/z calcd 304.2, found 304.2.

Synthesis of 1-ethyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP224) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.42 mmol) was dissolved in DMF (3 mL) and K2CO3 (220 mg, 1.6 mmol) and ethyl iodide (37 μL, 0.46 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.39 (s, 1H), 8.22–8.19 (m, 1H), 8.09 (d, J=8.5 Hz, 1H), 8.07–7.99 (m, 2H), 7.81 (dd, J=8.4, 1.7 Hz, 1H), 7.62–7.58 (m, 2H), 4.44 (q, J=7.2 Hz, 2H), 1.46 (t, J=7.2 Hz, 3H). ESI-MS (M+H)+ m/z calcd 290.1, found 290.2.

Synthesis of 1-cyclopentyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP228) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.42 mmol) was dissolved in DMF (3 mL) and K2CO3 (220 mg, 1.6 mmol) and cyclopentyl bromide (49.5 μL, 0.46 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.41 (s, 1H), 8.22–8.20 (m, 1H), 8.10 (d, J=8.5 Hz, 1H), 8.07–8.01 (m, 2H), 7.81 (dd, J=8.5, 1.7 Hz, 1H), 7.64–7.58 (m, 2H), 5.38–5.27 (m, 1H), 2.18–

N

N NH

N

NH2

N

N NN

NH2

228

N

N NH

N

NH2

N

N NN

NH2

224

79

2.04 (m, 4H), 1.96–1.89 (m, 2H), 1.77–1.69 (m, 2H). ESI-MS (M+H)+ m/z calcd 330.2, found 330.2.

Synthesis of 1-allyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP225) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and allyl iodide (23 μL, 0.25 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. ESI-MS (M+H)+ m/z calcd 302.1, found 302.2.

Synthesis of 2-(4-amino-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetamide (PP231) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and iodoacetamide (46 mg, 0.25 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.36 (s, 1H), 8.24–8.22 (m, 1H), 8.10 (d, J=8.4 Hz, 1H), 8.08–8.00 (m, 2H), 7.82 (dd, J=8.5, 1.6 Hz, 1H), 7.67 (br, 1H), 7.64–7.58 (m, 2H), 7.34 (br, 1H), 5.03 (s, 2H). ESI-MS (M+H)+ m/z calcd 319.1, found 319.2.

N

N NH

N

NH2

N

N NN

NH2

225

N

N NH

N

NH2

N

N NN

NH2

231NH2

O

80

Synthesis of 1-(cyclopropylmethyl)-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP165) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and cyclopropyl methyl bromide (22 μL, 0.25 mmol) were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.42 (s, 1H), 8.24–8.21 (m, 1H), 8.10 (d, J=8.4 Hz, 1H), 8.08–8.00 (m, 2H), 7.83 (dd, J=8.5, 1.5 Hz, 1H), 7.64–7.58 (m, 2H), 4.30 (d, J=7.1 Hz, 2H), 1.42–1.32 (m, 1H), 0.57–0.50 (m, 2H), 0.49–0.44 (m, 2H). ESI-MS (M+H)+ m/z calcd 316.2, found 316.2.

Synthesis of 1-isopentyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP232) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and isobutyl bromide were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.40 (s, 1H), 8.22–8.19 (m, 1H), 8.09 (d, J=8.5 Hz, 1H), 8.07–7.99 (m, 2H), 7.81 (dd, J=8.4, 1.7 Hz, 1H), 7.63–7.57 (m, 2H), 4.43 (d, J=7.2 Hz, 2H), 1.79 (td, J=7.4, 7.2 Hz, 2H), 1.58–1.51 (m, 1H), 0.94 (d, J=6.6 Hz, 6H). ESI-MS (M+H)+ m/z calcd 332.2, found 332.3.

N

N NH

N

NH2

N

N NN

NH2

165

N

N NH

N

NH2

N

N NN

NH2

232

N

N NH

N

NH2

N

N NN

NH2

167

81

Synthesis of 3-(naphthalen-2-yl)-1-(1-phenylallyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP167) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and 1-((E)-3-bromoprop-1-enyl)benzene were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. 1H NMR (DMSO–d6) δ 8.39 (s, 1H), 8.24–8.21 (m, 1H), 8.09 (d, J=8.6 Hz, 1H), 8.07–7.99 (m, 2H), 7.83 (dd, J=8.4, 1.8 Hz, 1H), 7.62–7.58 (m, 2H), 7.48–7.43 (m, 2H), 7.35–7.29 (m, 2H), 7.28–7.23 (m, 1H), 6.66 (d, J=15.8 Hz, 1H), 6.53 (td, J=15.9, 6.1 Hz, 1H), 5.22 (d, J=5.9 Hz, 2H). ESI-MS (M+H)+ m/z calcd 378.2, found 378.2.

Synthesis of 3-(naphthalen-2-yl)-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP168) 3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.21 mmol) was dissolved in DMF (1.5 mL) and K2CO3 (110 mg, 0.8 mmol) and propargylbromide were added. The reaction was heated to 60º C overnight, then cooled to RT and poured into water (30 mL). The precipitate was collected by filtration. ESI-MS (M+H)+ m/z calcd 300.1, found 300.2

N

N NH

N

NH2

N

N NN

NH2

168

82

Synthesis of 3-ethoxy-4-methoxybenzoyl chloride (ZK299) 3-ethoxy-4-methoxybenzoic acid (5 g, 25.5 mmol) was added to a solution of CH2Cl2 (40 mL) and benzene (20 mL) in a flame-dried 150 mL round bottom flask. Anhydrous DMF (9 drops) was added and the solution was cooled on ice. Oxalyl chloride (11 mL, 128 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT for 90 minutes, then concentrated in vacuo yield an off-white solid. The solid was placed on a high-vacuum line for 2 hours, and then taken onto the next step without further characterization. Synthesis of 2-((3-ethoxy-4-methoxyphenyl)(hydroxy)methylene)malononitrile (ZK301) NaH (2.2 g, 56 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (1.85 g, 28 mmol) in THF (30 mL) on ice. 3-ethoxy-4-methoxybenzoyl chloride (25.5 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The ice was then removed and the reaction was allowed to proceed at RT for 60 min. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc. The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give an orange solid that was taken onto the next step without further characterization. Synthesis of 2-((3-ethoxy-4-methoxyphenyl)(methoxy)methylene)malononitrile (ZK302) 2-((3-ethoxy-4-methoxyphenyl)(hydroxy)methylene)malononitrile (25.5 mmol) and sodium bicarbonate (17 g, 204 mmol) were combined in a solution of 1,4-dioxane (48 mL) and water (8 mL). Dimethylsulphate (17 mL, 178 mmol) was slowly added and the reaction was heated to 80-90º C for 2 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with EtOAc (200 mL). The organic phases were combined, dried with MgSO4, and filtered to give a red oil. The oil was purified by silica gel chromatography (10% EtOAc/Hexanes, Rf ~ 0.1) to give a white solid (3.59 g, 54.5% yield over three steps). ESI-MS (M+H)+ m/z calcd 259.1, found 259.0.

OHO

OMeOEt

ClO

OMeOEt

O

OMeOEt

CN

CNMeO

OMeOEt

CN

CN

OMeOEt

NNNC

H2N

N

N NN

OEt

OMeNH2

299 301 302303

305

83

Synthesis of 5-amino-3-(3-ethoxy-4-methoxyphenyl)-1-isopropyl-1H-pyrazole-4-carbonitrile (ZK303) 2-((3-ethoxy-4-methoxyphenyl)(methoxy)methylene)malononitrile (200 mg, 0.78 mmol), isopropylhydrazine hydrogen chloride (86 mg, 0.78 mmol), and triethylamine (0.10 mL, 0.78 mmol) were combined in ethanol (5 mL) and heated to reflux for 90 minutes. The reaction was then cooled to RT, water was added and the aqueous phase was extracted three times with EtOAc. The organic phase was concentrated and carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 301.1, found 301.0 Synthesis of 3-(3-ethoxy-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP305) 5-amino-3-(3-ethoxy-4-methoxyphenyl)-1-isopropyl-1H-pyrazole-4-carbonitrile was dissolved in formamide (20 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT, water was added, and the precipitate was collected by filtration. The precipitate was then dissolved in CH2Cl2/MeOH and passed through a silica plug. The product was then lyophilized from benzene to yield an off-white solid (48 mg, 19% over two steps). 1H NMR (CDCl3) δ 8.37 (s, 1H), 7.23–7.21 (m, 2H), 7.01 (d, J=8.4 Hz, 1H), 5.47 (s, 2H), 5.17 (m, 1H), 4.18 (q, J=7.0 Hz, 2H), 3.94 (s, 3H), 1.60 (d, J=6.7 Hz, 6H), 1.51 (t, J=7.0 Hz, 3H).ESI-MS (M+H)+ m/z calcd 328.2, found 328.0.

Synthesis of 5-amino-3-(3-ethoxy-4-methoxyphenyl)-1-(2-hydroxyethyl)-1H-pyrazole-4-carbonitrile (ZK304) 2-((3-ethoxy-4-methoxyphenyl)(methoxy)methylene)malononitrile (200 mg, 0.78 mmol), 2-hydroxyethylhydrazine (0.056 mL, 0.78 mmol), and triethylamine (0.10 mL, 0.78 mmol) were combined in ethanol (5 mL) and heated to reflux for 90 minutes. The reaction was then cooled to RT, water was added and the aqueous phase was extracted three times with EtOAc, CH2Cl2, and CHCl3. The organic phase was concentrated and carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 303.1, found 303.0. 2-(4-amino-3-(3-ethoxy-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethanol (PP306) 5-amino-3-(3-ethoxy-4-methoxyphenyl)-1-(2-hydroxyethyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (20 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT, water was added, and the precipitate was collected by filtration. The precipitate was then dissolved in CH2Cl2/MeOH and passed through a silica plug. The product was then lyophilized from benzene to yield a brown solid (6.4 mg, 2.5% over two steps). ESI-MS (M+H)+ m/z calcd 330.1, found 330.0.

MeO

OMeOEt

CN

CN

OMeOEt

NNNC

H2N

N

N NN

OEt

OMeNH2

302 OHOH304 306

84

Synthesis of 3-(benzyloxy)-4-methoxybenzoic acid (ZK296) Methyl 3-hydroxy-4-methoxybenzoate (5.064 g, 27.8 mmol) was dissolved in acetone (200 mL) in a flame-dried 500 mL round bottom flask. K2CO3 (25 g) and benzyl bromide (6.8 mL, 55 mmol) were added and the reaction was heated to reflux overnight. The next day the reaction was filtered and concentrated in vacuo. The residue was dissolved in MeOH (200 mL) and 5M NaOH (100 mL) and heated to reflux (3 hr). The reaction was then cooled to RT, the product precipitated by addition of 2N HCl, and the product collected by filtration to yield a white powder (6.13 g, 85%) that was taken onto the next step without further characterization. Synthesis of 3-(benzyloxy)-4-methoxybenzoyl chloride (ZK307) 3-(benzyloxy)-4-methoxybenzoic acid (6.13 g, 23.8 mmol) was added to CH2Cl2 (40 mL) in a flame-dried 150 mL round bottom flask. Anhydrous DMF (10 drops) was added and the solution was cooled on ice. Oxalyl chloride (10.3 mL, 118 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT for 90 minutes, then concentrated in vacuo yield an off-white solid. The solid was placed on a high-vacuum line overnight, and then taken onto the next step without further characterization. Synthesis of 2-((4-methoxy-3-benzyloxy)(hydroxy)methylene)malononitrile (ZK312) NaH (2.1 g, 52 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (1.7 g, 26 mmol) in THF (30 mL) on ice. 3-(benzyloxy)-4-methoxybenzoyl chloride (23.8 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 30 min and then allowed to proceed at RT for 60 min. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc (100 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give an red solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 307.1, found 307.0.

OMeOH

OMeO

OMeOBn

ONC

CN

OMeOBn

OMeNC

CN

OMe

OBn

NH

NNC

H2NN

N NH

N

OMe

OBnNH2

OMeOBn

OHO

OMeOBn

OCl

296 307 312 313

317319

85

Synthesis of 2-((3-(benzyloxy)-4-methoxyphenyl)(methoxy)methylene)malononitrile (ZK313) 2-((3-benzyloxy-4-methoxyphenyl)(hydroxy)methylene)malononitrile (23.8 mmol) and sodium bicarbonate (16 g, 190 mmol) were combined in a solution of 1,4-dioxane (50 mL) and water (10 mL). Dimethylsulphate (15.8 mL, 166 mmol) was slowly added and the reaction was heated to 80-90º C for 2 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with diethyl ether (200 mL). The organic phases were combined, dried with MgSO4, and filtered to give a red oil. The oil was purified by silica gel chromatography twice (10% EtOAc/Hexanes followed by 20% EtOAc/Hexanes) to give a white solid. ESI-MS (M+H)+ m/z calcd 321.1, found 321.0. Synthesis of 5-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazole-4-carbonitrile (ZK317) 2-((3-(benzyloxy)-4-methoxyphenyl)(methoxy)methylene)malononitrile (1 g, 3.1 mmol) and hydrazine monohydrate (0.17 mL, 3.5 mmol) were combined in ethanol (20 mL) and heated to reflux for 90 minutes. The reaction was then cooled to RT and allowed to stand overnight. The following day the product was collected as a yellow precipitate and taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 321.1, found 321.0. Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (ZK319) 5-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (10 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT and added to water (100 mL). The precipitate was collected by filtration, dissolved in CH2Cl2/MeOH and passed through a silica plug. ESI-MS (M+H)+ m/z calcd 348.1, found 348.0.

86

OEtOMe

OHO

OEtOMe

OCl

OEtOMe

ONC

CN

OEtOMe

OMeNC

CN

OEt

OMe

NH

NNC

H2N

N

N NH

N

OEt

OMeNH2

ZK308 ZK338 ZK339

ZK340PP341 Synthesis of 4-ethoxy-3-methoxybenzoyl chloride (ZK308) 4-ethoxy-3-methoxybenzoic acid (1.0 g, 5.1 mmol) was added to CH2Cl2 (~10 mL) in a flame-dried 25 mL round bottom flask. Anhydrous DMF (5 drops) was added and the solution was cooled on ice. Oxalyl chloride (2.17 mL, 25 mmol) was added dropwise, and the reaction was then allowed to proceed overnight at RT. The reaction was concentrated in vacuo, dissolved in CH2Cl2 and reconcentrated twice in vacuo, and placed on a high-vacuum line overnight. The off-white solid was then taken onto the next step without further characterization. Synthesis of 2-((4-ethoxy-3-methoxyphenyl)(hydroxy)methylene)malononitrile (ZK338) NaH (0.65 g, 16 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (0.50 g, 7.6 mmol) in THF (30 mL) on ice. 4-ethoxy-3-methoxybenzoyl chloride (5.1 mmol) was dissolved in THF (20 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 30 min and then allowed to proceed at RT for 60 min. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc (100 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give an yellow solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 245.1, found 245.0 Synthesis of 2-((4-ethoxy-3-methoxyphenyl)(methoxy)methylene)malononitrile (ZK339) 2-((4-ethoxy-3-methoxyphenyl)(hydroxy)methylene)malononitrile (5.1 mmol) and sodium bicarbonate (3.0 g, 36 mmol) were combined in a solution of 1,4-dioxane (16 mL) and water (2 mL). Dimethylsulphate (3.9 mL, 41 mmol) was slowly added and the reaction was heated to 80-90º C for 2 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried with MgSO4, and filtered to give an oil. The oil was purified by silica gel chromatography (15% EtOAc/Hexanes) to yield a white solid (935 mg, 77.5% yield over three steps). ESI-MS (M+H)+ m/z calcd 259.1, found 259.0.

87

Synthesis of 5-amino-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazole-4-carbonitrile (ZK340) 2-((4-ethoxy-3-methoxyphenyl)(methoxy)methylene)malononitrile (935 mg, 3.6 mmol) and hydrazine monohydrate (0.192 mL, 3.96 mmol) were combined in ethanol (10 mL) and heated to reflux for 90 minutes. The reaction was then cooled to RT, concentrated in vacuo, and taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 259.1, found 259.1 Synthesis of 3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP341) 5-amino-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazole-4-carbonitrile (~1 g) was dissolved in formamide (5 mL) in a 25 mL round bottom flask and heated to 180º C overnight. The next day the reaction was cooled to RT and a grey solid was collected by filtration. The precipitate was purified by silica gel chromatography (2% MeOH/CH2Cl2). 1H NMR (2:1 CDCl3:CD3OD) δ 8.31 (s, 1H), 8.11 (br, 2H), 7.26–7.20 (m, 2H), 7.07 (d, J=8.0 Hz, 1H), 4.21 (m, 2H, obscured by water peak), 3.97 (s, 3H), 1.53 (t, J=7.0 Hz, 3H). ESI-MS (M+H)+ m/z calcd 286.1, found 286.1.

Synthesis of 3-(benzyloxy)-4-methylbenzoic acid (ZK295) 3-hydroxy-4-methylbenzoic acid (5.0 g, 33 mmol) was dissolved in acetone (200 mL) in a flame-dried 500 mL round bottom flask. K2CO3 (20 g) and benzyl bromide (16.0 mL, 131 mmol) were added and the reaction was heated to reflux overnight. The next day the reaction was filtered and concentrated in vacuo. The residue was dissolved in MeOH (200 mL) and 5M NaOH (100 mL) and heated to reflux (3 hr). The reaction was then cooled to RT and concentrated in vacuo. Water (300 mL) was added and the aqueous phase was washed twice with hexanes. The aqueous phase was then acidified by addition of 2N HCl until a thick white precipitate formed. The product was collected

MeOH

OHO

MeOBn

ONC

CN

MeOBn

OMeNC

CN

Me

OBn

NH

NNC

H2NN

N NH

N

Me

OBnNH2

MeOBn

OHO

MeOBn

OCl

295 309 314 315

316318

88

by filtration to yield a white powder (7.0 g, 99%) that was taken onto the next step without further characterization. Synthesis of 3-(benzyloxy)-4-methylbenzoyl chloride (ZK309) 3-(benzyloxy)-4-methylbenzoic acid (32.8 mmol) was added to CH2Cl2 (50 mL) in a flame-dried 150 mL round bottom flask. Anhydrous DMF (12 drops) was added and the solution was cooled on ice. Oxalyl chloride (14.2 mL, 164 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT overnight, concentrated in vacuo, and then reconcentrated twice from CH2Cl2 to yield an off-white solid. The solid was placed on a high-vacuum line overnight, and then taken onto the next step without further characterization. Synthesis of 2-((4-methyl-3-benzyl)(hydroxy)methylene)malononitrile (ZK314) NaH (2.9 g, 72 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (2.4 g, 36 mmol) in THF (50 mL) on ice. 3-(benzyloxy)-4-methylbenzoyl chloride (32.8 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 60 min and then allowed to proceed at RT for 60 min. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc (100 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give an brown solid that was taken onto the next step without further characterization. Synthesis of 2-((3-(benzyloxy)-4-methyl)(methoxy)methylene)malononitrile (ZK315) 2-((4-methyl-3-benzyloxy)(hydroxy)methylene)malononitrile (32.8 mmol) and sodium bicarbonate (22 g, 262 mmol) were combined in a solution of 1,4-dioxane (50 mL) and water (10 mL). Dimethylsulphate (22 mL, 230 mmol) was slowly added and the reaction was heated to 80-90º C for 4 hours. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with diethyl ether (200 mL). The organic phases were combined, dried with MgSO4, and filtered to give an oil. The oil was purified by silica gel chromatography (10% EtOAc/Hexanes) to give a white solid (1.94 g, 19.5% yield over three steps). ESI-MS (M+H)+ m/z calcd 305.1, found 305.0. Synthesis of 5-amino-3-(3-(benzyloxy)-4-methyl)-1H-pyrazole-4-carbonitrile (ZK316) 2-((3-(benzyloxy)-4-methyl)(methoxy)methylene)malononitrile (1.94 g, 6.4 mmol) and hydrazine monohydrate (0.31 mL, 6.4 mmol) were combined in ethanol (20 mL) and heated to reflux for 90 minutes. The reaction was then cooled to RT and allowed to stand at RT overnight. The product precipitated overnight and was collected as a white solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 305.1, found 305.0 Synthesis of 3-(3-(benzyloxy)-4-methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP318) 5-amino-3-(3-(benzyloxy)-4-methyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (10 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT and added to water (100 mL). The precipitate was collected by filtration to yield a grey solid, which was dissolved in CH2Cl2/MeOH and passed through a silica plug (5% MeOH/CH2Cl2) to yield a white solid (470 mg, 22.2% yield over two steps). 1H NMR (DMSO–d6) δ 13.53 (s, 1H), 8.21 (s, 1H), 7.50–7.48 (m, 2H), 7.44–7.39 (m, 2H), 7.35–7.29 (m, 3H), 7.17 (dd, J=7.5, 1.4 Hz, 1H), 5.21 (s, 2H), 2.29 (s, 3H). ESI-MS (M+H)+ m/z calcd 332.2, found 332.0.

89

Synthesis of 5-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methylphenol (PP320)

3-(3-(benzyloxy)-4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in formic acid (1 mL). Concentrated HCl (0.1 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in MeOH/CH2Cl2 and concentrated in vacuo to yield an off-white solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (13 mg, 35.7% yield). 1H NMR (2:1 CDCl3:CD3OD) δ 8.30 (s, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.07–7.03 (m, 2H), 2.31 (s, 3H). ESI-MS (M+H)+ m/z calcd 242.1, found 242.0.

Synthesis of 5-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP321)

3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in formic acid (1 mL). Concentrated HCl (0.1 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in MeOH/CH2Cl2 and concentrated in vacuo to yield a yellow solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (11 mg, 29.7% yield). 1H NMR (2:1 CDCl3:CD3OD) δ 8.31 (s, 1H), 7.17–7.12 (m, 2H), 7.07 (d, J=8.3 Hz, 1H), 3.99 (s, 3H). ESI-MS (M+H)+ m/z calcd 258.1, found 258.0.

N

N NN

OBn

Me

NH2

N

N NN

OH

Me

NH2

318 322 323

N

N NH

N

OBn

Me

NH2

N

N NH

N

OH

OMe

NH2

319 321

N

N NH

N

OBn

OMe

NH2

N

N NH

N

OH

Me

NH2

318 320

N

N NH

N

OBn

Me

NH2

90

Synthesis of 3-(3-(benzyloxy)-4-methylphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP322) 3-(3-(benzyloxy)-4-methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.3 mmol) was dissolved in DMF (1 mL). Potassium carbonate (166 mg, 1.2 mmol) and isopropyl bromide (0.95 mL, 1.0 mmol) were added and the reaction was heated to 80º C overnight. The reaction was then cooled to RT, poured into water, and the white precipitate collected by filtration. ESI-MS (M+H)+ m/z calcd 374.2, found 374.1. Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methylphenol (PP323) 3-(3-(benzyloxy)-4-methylphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (1 mL). Concentrated HCl (0.1 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in MeOH/CH2Cl2 and concentrated in vacuo to yield a solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (17 mg, 20% yield over two steps). 1H NMR (2:1 CDCl3:CD3OD) δ 8.28 (s, 1H), 7.29 (d, J=7.5 Hz, 1H), 7.09–7.04 (m, 2H), 5.18 (m, 1H), 2.31 (s, 3H), 1.61 (d, J=6.7 Hz, 6H). ESI-MS (M+H)+ m/z calcd 284.2, found 284.0.

Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP324) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.29 mmol) was dissolved in DMF (1 mL). Potassium carbonate (198 mg, 1.44 mmol) and isopropyl bromide (0.43 mL, 4.8 mmol) were added and the reaction was heated to 80º C overnight. The reaction was then cooled to RT, poured into water, and the white precipitate collected by filtration. ESI-MS (M+H)+ m/z calcd 390.2, found 390.0. Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP325) 3-(3-(benzyloxy)-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 30 min. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo to yield a solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (25.5 mg, 29.6% yield over two steps). 1H NMR (DMSO–d6) δ 9.36 (br, 1H), 8.34 (s, 1H), 7.10–7.06 (m, 3H), 5.07 (m, 1H), 3.84 (s, 3H), 1.49 (d, J=6.7 Hz, 6H). ESI-MS (M+H)+ m/z calcd 300.1, found 300.0.

N

N NN

OBn

OMe

NH2

N

N NN

OH

OMe

NH2

319 324 325

N

N NH

N

OBn

OMe

NH2

91

Synthesis of 1-sec-butyl-3-(3-(benzyloxy)-4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP326) 3-(3-(benzyloxy)-4-methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (110 mg, 0.33 mmol) was dissolved in DMF (2 mL). Potassium carbonate (229 mg, 1.6 mmol) and 2-bromobutane (0.17 mL, 1.6 mmol) were added and the reaction was heated to 80º C for 3 hr. The reaction was then cooled to RT, poured into water, and the precipitate collected by filtration. ESI-MS (M+H)+ m/z calcd 388.2, found 388.1. Synthesis of 5-(1-sec-butyl-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methylphenol (PP327) 1-sec-butyl-3-(3-(benzyloxy)-4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo to yield a solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (3.8 mg, 9.8% yield over two steps). 1H NMR (DMSO–d6) δ 9.65 (br, 1H), 8.33 (s, 1H), 7.24 (d, J=7.7 Hz, 1H), 7.10 (d, J=1.6 Hz, 1H), 6.99 (dd, J=7.6, 1.6 Hz, 1H), 4.86–4.80 (m, 1H), 2.20–2.18 (m, 3H), 1.99–1.81 (m, 2H), 1.47 (d, J=6.7 Hz, 3H), 0.71 (d, J=7.4 Hz, 3H). ESI-MS (M+H)+ m/z calcd 298.2, found 298.1.

Synthesis of 3-(3-(benzyloxy)-4-methylphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP332) 3-(3-(benzyloxy)-4-methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (36 mg, 0.11 mmol) was dissolved in DMF (2 mL). Potassium carbonate (100 mg) and methyl iodide (0.008 mL, 0.12 mmol) were added and the reaction was allowed to proceed at RT for 30 min. The reaction was then poured into water, and the precipitate collected by filtration. ESI-MS (M+H)+ m/z calcd 346.2, found 346.0.

N

N NN

OBn

Me

NH2

N

N NN

OH

Me

NH2

Me Me318 332 333

N

N NH

N

OBn

Me

NH2

N

N NN

OBn

Me

NH2

N

N NN

OH

Me

NH2

318 326 327

N

N NH

N

OBn

Me

NH2

92

Synthesis of 5-(4-amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methylphenol (PP333) 3-(3-(benzyloxy)-4-methylphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 30 min. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo to yield a solid. The solid was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.3 mg, 27.7% yield over two steps). 1H NMR (DMSO–d6) δ 9.66 (br, 1H), 8.33 (s, 1H), 7.24 (d, J=7.6 Hz, 1H), 7.09 (d, J=1.6 Hz, 1H), 6.99 (dd, J=7.6, 1.6 Hz, 1H), 3.96 (s, 3H), 2.19 (s, 3H). ESI-MS (M+H)+ m/z calcd 256.1, found 256.1.

Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP336) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (25 mg, 0.07 mmol) was dissolved in DMF (1 mL). Potassium carbonate (120 mg) and methyl iodide (0.005 mL, 0.08 mmol) were added and the reaction was stirred at RT for 30 min. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. Synthesis of 5-(4-amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP337) 3-(3-(benzyloxy)-4-methoxyphenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (1.2 mg, 6.1% yield over two steps). ESI-MS (M+H)+ m/z calcd 272.1, found 272.0.

N

N NH

N

OBn

OMe

NH2

N

N NN

OBn

OMe

NH2

N

N NN

OH

OMe

NH2

Me Me319 336 337

93

Synthesis of 3-(4-ethoxy-3-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP343) 3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.18 mmol) was was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and 2-iodopropane (0.063 mL, 0.63 mmol) were added and the reaction was allowed to proceed at RT for 2 hr. Water was added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (3.2 mg, 5.4% yield). ESI-MS (M+H)+ m/z calcd 328.2, found 328.1.

Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP346) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.16 mmol) was dissolved in DMF (2 mL). Potassium carbonate (250 mg) and ethyl iodide (0.1 mL) were added and the reaction was stirred at 60º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. Synthesis of 5-(4-amino-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP347) 3-(3-(benzyloxy)-4-methoxyphenyl)-1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (3 mL). Concentrated HCl (0.3 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (9 mg, 21.9% yield over two steps). 1H NMR (CDCl3) δ 11.68 (br, 1H), 8.22 (s, 1H), 7.22 (d, J=2.1 Hz, 1H), 7.11 (dd, J=8.2, 2.1 Hz, 1H), 7.02 (d, J=8.2 Hz, 1H), 6.34 (br, 1H), 4.52

N

N NH

N

OEt

OMeNH2

341

N

N NN

OEt

OMeNH2

343

N

N NH

N

OMe

OBnNH2

319

N

N NN

OMe

OBnNH2

N

N NN

OMe

OHNH2

346 347

94

(q, J=7.3 Hz, 2H), 3.99 (s, 3H), 1.58 (t, J=7.3 Hz, 3H). ESI-MS (M+H)+ m/z calcd 286.1, found 286.0.

Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP348) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (33 mg, 0.095 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and cyclopentyl bromide (0.1 mL, 1.0 mmol) were added and the reaction was allowed to proceed at 90º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. ESI-MS (M+H)+ m/z calcd 416.2, found 416.0. Synthesis of 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP349) 3-(3-(benzyloxy)-4-methoxyphenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (1 mL). Concentrated HCl (0.1 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (7.1 mg, 23% yield over two steps). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.24 (s, 1H), 7.12 (dd, J=8.2, 2.1 Hz, 1H), 7.01 (d, J=8.3 Hz, 1H), 5.34–5.26 (m, 1H), 3.98 (s, 3H), 2.23–2.14 (m, 4H), 2.06–1.96 (m, 2H), 1.81–1.70 (m, 2H). ESI-MS (M+H)+ m/z calcd 326.2, found 326.0.

N

N NH

N

OMe

OBnNH2

319

N

N NN

OMe

OBnNH2

N

N NN

OMe

OHNH2

348 349

95

Synthesis of 3-(benzyloxy)-4-chlorobenzoic acid (ZK351) 4-chloro-3-hydroxybenzoic acid (3.00 g, 17.4 mmol) was dissolved in acetone (150 mL) in a flame-dried 250 mL round bottom flask. K2CO3 (20 g) and benzyl bromide (8.5 mL, 70 mmol) were added and the reaction was heated to reflux overnight. The next day the reaction was filtered and concentrated in vacuo. The residue was dissolved in MeOH (200 mL) and 5M NaOH (100 mL) and heated to reflux (2.5 hr). The reaction was then cooled to RT, water was added, and the aqueous phase was acidified by addition of 2N HCl until a thick white precipitate formed. The product was collected by filtration to yield a white powder (4.5 g, quant.) that was taken onto the next step without further characterization. Synthesis of 3-(benzyloxy)-4-chlorobenzoyl chloride (ZK352) 3-(benzyloxy)-4-chlorobenzoic acid (18 mmol) was added to CH2Cl2 (50 mL). Anhydrous DMF (10 drops) was added and the solution was cooled on ice. Oxalyl chloride (15.6 mL, 180 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT for 3 h, concentrated in vacuo, and then reconcentrated twice from CH2Cl2. The solid was placed on a high-vacuum line overnight, and then taken onto the next step without further characterization. Synthesis of 2-((3-(benzyloxy)-4-chlorophenyl)(hydroxy)methylene)malononitrile (ZK353) NaH (2.9 g, 72 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (1.8 g, 27 mmol) in THF (50 mL) on ice. 3-(benzyloxy)-4-chlorobenzoyl chloride (18 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 60 min and then allowed to proceed at RT for 60 min. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc (100 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give a solid that was taken onto the next step without further characterization.

ClOH

OHO

ClOBn

NC

CN

ClOBn

OMeNC

CN

Cl

OBn

NH

NNC

H2NN

N NH

N

Cl

OBnNH2

ClOBn

OHO

ClOBn

OCl

351 352 353 354

355356

OH

96

Synthesis of 2-((3-(benzyloxy)-4-chlorophenyl)(methoxy)methylene)malononitrile (ZK354) 2-((3-(benzyloxy)-4-chlorophenyl)(hydroxy)methylene)malononitrile (18 mmol) and sodium bicarbonate (10.6 g, 126 mmol) were combined in a solution of 1,4-dioxane (50 mL) and water (10 mL). Dimethylsulphate (14 mL, 144 mmol) was slowly added and the reaction was heated to 80º C for 1 h. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried with MgSO4, filtered, and concentrated in vacuo. The resultant oil was purified by silica gel chromatography (20% EtOAc/Hexanes) to give a white solid that was taken onto the next step without further characterization. Synthesis of 5-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazole-4-carbonitrile (ZK355) 2-((3-(benzyloxy)-4-chlorophenyl)(methoxy)methylene)malononitrile and hydrazine monohydrate (1.0 mL, 20 mmol) were combined in ethanol (20 mL) and allowed to proceed at RT for 90 minutes. The reaction was then concentrated in vacuo, and taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 325.1, found 325.4 Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP356) 5-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (20 mL) and heated to 170º C overnight. The next day the reaction was cooled to RT and water was added (40 mL). The precipitate was collected by filtration to yield an off-white solid. (2.02 g, 32.8% yield over five steps). ESI-MS (M+H)+ m/z calcd 352.1, found 352.4.

Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP357) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and isopropyl iodide (0.044 mL, 0.45 mmol) were added and the reaction was stirred at RT for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. ESI-MS (M+H)+ m/z calcd 394.1, found 394.4.

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

357

N

N NN

Cl

OHNH2

358

97

Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP358) 3-(3-(benzyloxy)-4-chlorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (14.1 mg, 33.2% yield over two steps). ESI-MS (M+H)+ m/z calcd 304.1, found 304.4.

Synthesis of 1-cyclopentyl-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP359) 3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and cyclopentyl iodide (0.081 mL, 0.69 mmol) were added and the reaction was stirred at RT for 2 hr. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (7.0 mg, 11%). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.16–7.11 (m, 2H), 7.01 (d, J=8.0 Hz, 1H), 5.34–5.27 (m, 1H), 4.18 (q, J=7.0 Hz, 2H), 3.94 (s, 3H), 2.23–2.15 (m, 4H), 2.05–1.96 (m, 2H), 1.81–1.73 (m, 2H), 1.52 (t, J=7.0 Hz, 3H). ESI-MS (M+H)+ m/z calcd 354.2, found 354.1.

Synthesis of 1-allyl-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP361) 3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.18 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and allyl iodide

N

N NH

N

OEt

OMeNH2

341

N

N NN

OEt

OMeNH2

359

N

N NH

N

OEt

OMeNH2

341

N

N NN

OEt

OMeNH2

361

98

(0.032 mL, 0.35 mmol) were added and the reaction was stirred at RT for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (9.0 mg, 16%). 1H NMR (CDCl3) δ 11.68 (br, 1H), 8.24 (s, 1H), 7.17 (d, J=2.0 Hz, 1H), 7.13 (dd, J=8.1, 2.1 Hz, 1H), 7.02 (d, J=8.2 Hz, 1H), 6.37 (br, 1H), 6.09 (m, 1H), 5.35–5.30 (m, 2H), 5.08 (td, J=5.9, 1.3 Hz, 2H), 4.18 (q, J=7.0 Hz, 2H), 4.00–3.90 (m, 3H), 1.53 (t, J=7.0 Hz, 3H). ESI-MS (M+H)+ m/z calcd 326.2, found 326.0.

Synthesis of 1-cyclohexyl-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP362) 1-cyclohexyl-3-(4-ethoxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.18 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and cyclohexyl iodide (0.42 mL, 5.6 mmol) were added and the reaction was allowed to proceed at 90º C overnight. The reaction was cooled to RT, water was added, and the reaction extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (9.7 mg, 15%). ESI-MS (M+H)+ m/z calcd 368.2, found 368.1.

N

N NH

N

OEt

OMeNH2

341

N

N NN

OEt

OMeNH2

362

99

Synthesis of 3-(benzyloxy)-4-fluorobenzoic acid (ZK297) 4-fluoro-3-hydroxybenzoic acid (4.8 g, 32 mmol) was added to acetone (200 mL) in a flame-dried 500 mL round bottom flask. K2CO3 (25 g) and benzyl bromide (15.4 mL mL, 126 mmol) were added and the reaction was heated to reflux overnight. The next day the reaction was filtered and concentrated in vacuo. The residue was dissolved in MeOH (200 mL) and 5M NaOH (100 mL) and heated to reflux (3 hr). The reaction was then cooled to RT, the product precipitated by addition of 2N HCl, and the product collected by filtration to yield a white powder (6.11 g, 79%) that was taken onto the next step without further characterization. Synthesis of 3-(benzyloxy)-4-fluorobenzoyl chloride (ZK363) 3-(benzyloxy)-4-fluorobenzoic acid (6.1 g, 25 mmol) was added to CH2Cl2 (50 mL). Anhydrous DMF (10 drops) was added and the solution was cooled on ice. Oxalyl chloride (21.5 mL, 250 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT overnight, concentrated in vacuo, and then reconcentrated twice from CH2Cl2. The solid was placed on a high-vacuum line overnight, and then taken onto the next step without further characterization. Synthesis of 2-((3-(benzyloxy)-4-fluorophenyl)(hydroxy)methylene)malononitrile (ZK364) NaH (4.0 g, 100 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (2.5 g, 38 mmol) in THF (50 mL) on ice. 3-(benzyloxy)-4-fluorobenzoyl chloride (25 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 1 h and then allowed to proceed at RT for 4 hours. 1N HCl (200 mL) was added and the solution was extracted three times with EtOAc (200 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give a tan solid that was taken onto the next step without further characterization.

FOBn

OCl

FOBn

ONC

CN

FOBn

OMeNC

CN

F

OBn

NH

NNC

H2N

N

N NH

N

F

OBnNH2

364

365366367

FOBn

OHO

297

FOH

OHO

363

100

Synthesis of 2-((3-(benzyloxy)-4-fluorophenyl)(methoxy)methylene)malononitrile (ZK365) 2-((3-(benzyloxy)-4-fluorophenyl)(hydroxy)methylene)malononitrile (25 mmol) and sodium bicarbonate (14.7 g, 175 mmol) were combined in a solution of 1,4-dioxane (80 mL) and water (10 mL). Dimethylsulphate (19 mL, 200 mmol) was slowly added and the reaction was heated to 80º C for 1 h. The reaction was cooled to RT, water was added, and the aqueous phase extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried with MgSO4, filtered, and concentrated in vacuo. The resultant oil was purified by silica gel chromatography (20% EtOAc/Hexanes) to yield a white solid (7.0 g, 72% over three steps). Synthesis of 5-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazole-4-carbonitrile (ZK366) 2-((3-(benzyloxy)-4-fluorophenyl)(methoxy)methylene)malononitrile (7.0 g, 2.3 mmol) and hydrazine monohydrate (1.33 mL, 27.5 mmol) were combined in ethanol (50 mL) and allowed to proceed at RT for 1 hour. The reaction was then concentrated in vacuo to yield a yellow solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 309.1, found 309.4 Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP367) 5-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (15 mL) and heated to 170º C overnight. The next day the reaction was cooled to RT and a thick precipitate formed. The precipitate was washed with water and purified by silica gel chromatography (gradient of 1-5% MeOH in CH2Cl2) to yield a pale solid (3.424 g, 45% yield over two steps). ESI-MS (M+H)+ m/z calcd 336.1, found 336.4.

Synthesis of tert-butyl 3-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (PP368) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 3-(bromomethyl)piperidine-1-carboxylate (167 mg, 0.6 mmol) were added and the reaction was heat to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

N NH

O O

368 369

101

concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 549.2, found 549.5. Synthesis of 5-(4-amino-1-((piperidin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP369) tert-butyl 3-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (9.7 mg, 19% yield over two steps). ESI-MS (M+H)+ m/z calcd 359.1, found 359.5.

Synthesis of tert-butyl 4-((4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (PP376) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (89 mg, 0.45 mmol) were added and the reaction was heat to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 533.3, found 533.2. Synthesis of 5-(4-amino-1-((piperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP377) tert-butyl 4-((4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (12 mg, 24% yield over two steps). ESI-MS (M+H)+ m/z calcd 343.2, found 343.1.

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

N

N NN

F

OHNH2

N HN

376 377

OO

102

Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP370) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and cyclopentyl iodide (0.052 mL, 0.45 mmol) were added and the reaction was stirred at RT for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo. Synthesis of 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP371) 3-(3-(benzyloxy)-4-chlorophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (18.4 mg, 39.3% yield over two steps). 1H NMR (CDCl3) δ 8.23 (s, 1H), 7.52 (d, J=8.2 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.15 (dd, J=8.2, 2.0 Hz, 1H), 5.34–5.30 (m, 1H), 2.22–2.15 (m, 4H), 2.03–1.99 (m, 2H), 1.79–1.75 (m, 2H). ESI-MS (M+H)+ m/z calcd 330.1, found 330.4.

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP378) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and cyclopentyl iodide (0.052 mL, 0.45 mmol) were added and the reaction was stirred at RT for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2.

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

N

N NN

F

OHNH2

378 379

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

370 371

103

The organic phases were combined and concentrated in vacuo. ESI-MS (M+H)+ m/z calcd 404.2, found 404.1. Synthesis of 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP379) 3-(3-(benzyloxy)-4-fluorophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (16 mg, 34% yield over two steps). ESI-MS (M+H)+ m/z calcd 314.1, found 314.1.

Synthesis of tert-butyl 4-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate (PP384) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 4-(bromomethyl)piperidine-1-carboxylate (89 mg, 0.45 mmol) were added and the reaction was heat to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. Synthesis of 5-(4-amino-1-((piperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP385) tert-butyl 4-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (34 mg, 67% yield over two steps). ESI-MS (M+H)+ m/z calcd 359.1, found 359.1.

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

N HN

384 385O

O

104

Synthesis of tert-butyl 2-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate (PP386) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 2-(bromomethyl)pyrrolidine-1-carboxylate (82 mg, 0.45 mmol) were added and the reaction was heat to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 535.2, found 535.1. Synthesis of 5-(4-amino-1-((pyrrolidin-2-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP387) tert-butyl 2-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (19.7 mg, 40.2% yield over two steps). ESI-MS (M+H)+ m/z calcd 345.1, found 345.1.

Synthesis of tert-butyl 3-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate (ZK388)

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

386 387

N NHO

O

N

N NH

N

Cl

OBnNH2

356

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

388 389

N NH

OO

105

3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 3-(bromomethyl)pyrrolidine-1-carboxylate (82 mg, 0.45 mmol) were added and the reaction was heat to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 535.2, found 535.1. Synthesis of 5-(4-amino-1-((pyrrolidin-3-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP389) tert-butyl 3-((4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyrrolidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (14.5 mg, 29.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 345.1, found 345.0.

Synthesis of tert-butyl 4-(2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)piperidine-1-carboxylate (PP390) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 4-(2-bromoethyl)piperidine-1-carboxylate (131 mg, 0.45 mmol) were added and the reaction was heat to 80º C for 3 hours. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 563.3, found 563.1. Synthesis of 5-(4-amino-1-(2-(piperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP391) tert-butyl 4-(2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)piperidine-1-carboxylate was dissolved in formic acid (2 mL). Concentrated HCl (0.2 mL) was added and the reaction was heated to reflux for 1 h. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

N NH

O O356

390 391

106

(MeCN:H2O:0.1% TFA) to yield a white solid (21 mg, 40% yield over two steps). ESI-MS (M+H)+ m/z calcd 373.2, found 373.1.

Synthesis of (S)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (ZK393) (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (548 mg, 2.93 mmol) was dissolved in CH2Cl2 (4 mL) and triethylamine (2.5 mL, 17.5 mmol). The reaction was cooled on ice and methanesulfonyl chloride (1.1 mL, 14 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (2% MeOH/CH2Cl2) to yield a clear oil (481 mg, 62%).

Synthesis of (R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (ZK395) (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (1 g, 5.3 mmol) was dissolved in CH2Cl2 (4 mL) and triethylamine (2.77 mL, 20 mmol). The reaction was cooled on ice and methanesulfonyl chloride (1.15 mL, 15 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (2% MeOH/CH2Cl2) to yield a clear oil (784 mg, 55%).

Synthesis of 1-(tert-butoxycarbonyl)azetidin-3-yl methanesulfonate (ZK397) tert-butyl 3-hydroxyazetidine-1-carboxylate (1 g, 5.7 mmol) was dissolved in CH2Cl2 (10 mL) and triethylamine (3.2 mL, 23 mmol). The reaction was cooled on ice and methanesulfonyl chloride (1.3 mL, 17 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (2% MeOH/CH2Cl2) to yield a clear oil (1.2 g, 85%).

N OHO

ON OMs

O

O

393

N OHO

ON OMs

O

O

395

NHOO

ONMsO

O

O

397

107

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-((5-methyl-2-phenyl-2H-1,2,3-triazol-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP398) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and 4-(bromomethyl)-5-methyl-2-phenyl-2H-1,2,3-triazole (55 mg, 0.22 mmol) were added and the reaction was heat to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 507.2, found 507.1. Synthesis of 5-(4-amino-1-((5-methyl-2-phenyl-2H-1,2,3-triazol-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP399) 3-(3-(benzyloxy)-4-fluorophenyl)-1-((5-methyl-2-phenyl-2H-1,2,3-triazol-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (7.4 mg, 12% yield over two steps). 1H NMR (2:1 CDCl3:CD3OD) δ 8.38 (s, 1H), 7.98–7.95 (m, 2H), 7.47–7.42 (m, 2H), 7.34–7.30 (m, 1H), 7.24–7.18 (m, 2H), 7.10–7.07 (m, 1H), 5.75 (s, 2H), 2.40 (s, 3H). ESI-MS (M+H)+ m/z calcd 417.2, found 417.0.

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-((4-phenyloxazol-5-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP400)

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

NN

N

N

N NN

F

OHNH2

NN

N

398 399

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

N

O

N

N NN

F

OHNH2

N

O

400 401

108

3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and 5-(bromomethyl)-4-phenyloxazole (52 mg, 0.22 mmol) were added and the reaction was heat to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 493.2, found 493.1. Synthesis of 5-(4-amino-1-((4-phenyloxazol-5-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP401) 3-(3-(benzyloxy)-4-fluorophenyl)-1-((4-phenyloxazol-5-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.7 mg, 9.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 403.1, found 403.0.

Synthesis of (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate (PP402) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.32 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and (S)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (0.32 mL, 1.2 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 505.2, found 505.1. Synthesis of 5-(4-amino-1-((S)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP403) (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2.5 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (44 mg, 47% yield over two steps). ESI-MS (M+H)+ m/z calcd 315.1, found 315.1.

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

N

N NN

F

OHNH2

402403

N NH

O

O

109

Synthesis of (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate (PP404) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.32 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and (R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (250 mg, 0.94 mmol) were added and the reaction was heated to 80º C for 4 hours. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 505.2, found 505.1. Synthesis of 5-(4-amino-1-((S)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP405) (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2.5 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (25 mg, 27% yield over two steps). ESI-MS (M+H)+ m/z calcd 315.1, found 315.1.

N

N NH

N

F

OBnNH2

367

N

N NN

F

OBnNH2

N

N NN

F

OHNH2

404405

N NH

O

O

N

N NH

N

OMe

OBnNH2

N

N NN

OMe

OBnNH2

N

N NN

OMe

OHNH2

406407

N NH

O

O319

110

Synthesis of (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate (PP406) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.29 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and (R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (250 mg, 0.94 mmol) were added and the reaction was heated to 80º C for 8 hours. Water was then added and the reaction as extracted four times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 517.3, found 517.1. Synthesis of 5-(4-amino-1-((S)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP407) (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.0 mg, 5.3% yield over two steps). ESI-MS (M+H)+ m/z calcd 327.2, found 327.1.

Synthesis of (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate (PP408) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.3 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and (R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yl methanesulfonate (250 mg, 0.94 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted four times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 521.2, found 521.1. Synthesis of 5-(4-amino-1-((S)-pyrrolidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP409) (S)-tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OBnNH2

N

N NN

Cl

OHNH2

408409

N NH

O

O356

111

(MeCN:H2O:0.1% TFA) to yield a white solid (23 mg, 24% yield over two steps). ESI-MS (M+H)+ m/z calcd 331.1, found 331.0.

Synthesis of tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidine-1-carboxylate (PP412) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.3 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 3-bromoazetidine-1-carboxylate (452 mg, 1.8 mmol) were added and the reaction was heated to 80º C for 48 hours. Water was then added and the reaction as extracted four times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 491.2, found 491.1. Synthesis of 5-(4-amino-1-(azetidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP413) tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (8.5 mg, 9.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 301.1, found 301.1.

Synthesis of 3-methylcyclopentyl methanesulfonate (ZK418) 3-methylcyclopentanol (1 g, 10 mmol) was dissolved in CH2Cl2 (10 mL) and triethylamine (5.5 mL, 40 mmol). The reaction was cooled on ice and methanesulfonyl chloride (2.3 mL, 30 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (10% EtOAc/Hexanes) to yield a clear oil (1.26 g, 82%).

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

412413

367 NHN

OO

Me OH Me OMs

418

112

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP420) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (100 mg, 0.3 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and 3-methylcyclopentyl methanesulfonate (220 mg, 1.2 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 418.2, found 418.1 Synthesis of 5-(4-amino-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP421) 3-(3-(benzyloxy)-4-fluorophenyl)-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (25 mg, 25.6% yield over two steps). ESI-MS (M+H)+ m/z calcd 328.2, found 328.1.

Synthesis of 3-(3-(benzyloxy)-4-methoxyphenyl)-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP422) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and 3-methylcyclopentyl methanesulfonate (220 mg, 1.2 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

420 421

367Me Me

N

N NH

N

OMe

OBnNH2

N

N NN

OMe

OHNH2

N

N NN

OMe

OBnNH2

422 423

319Me Me

113

three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 430.2, found 430.1 Synthesis of 5-(4-amino-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP423) 3-(3-(benzyloxy)-4-methoxyphenyl)-1-(3-methylcyclopentyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (8.7 mg, 18% yield over two steps). ESI-MS (M+H)+ m/z calcd 340.2, found 340.1.

Synthesis of tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidine-1-carboxylate (PP410) 3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.14 mmol) was dissolved in DMF (1 mL). Potassium carbonate (250 mg) and tert-butyl 3-bromoazetidine-1-carboxylate (300 mg, 1.2 mmol) were added and the reaction was heated to 80º C for 48 hours. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 503.2, found 503.1. Synthesis of 5-(4-amino-1-(azetidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-methoxyphenol (PP411) tert-butyl 3-(4-amino-3-(3-(benzyloxy)-4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azetidine-1-carboxylate was dissolved in formic acid (10 mL). Concentrated HCl (1 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (15.7 mg, 34.9% yield over two steps). ESI-MS (M+H)+ m/z calcd 313.1, found 313.1.

N

N NH

N

OMe

OBnNH2

N

N NN

OMe

OHNH2

N

N NN

OMe

OBnNH2

410

411

319 N NH

OO

114

Synthesis of (R)-tetrahydrofuran-3-yl methanesulfonate (ZK425) (R)-tetrahydrofuran-3-ol (1.0 g, 11 mmol) was dissolved in CH2Cl2 (20 mL) and triethylamine (9.4 mL, 86 mmol). The reaction was cooled on ice and methanesulfonyl chloride (2.6 mL, 34 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography to yield a clear oil (1.32 g, 70.4%).

Synthesis of (S)-tetrahydrofuran-3-yl methanesulfonate (ZK426) (S)-tetrahydrofuran-3-ol (1.0 g, 11 mmol) was dissolved in CH2Cl2 (20 mL) and triethylamine (9.4 mL, 86 mmol). The reaction was cooled on ice and methanesulfonyl chloride (2.6 mL, 34 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography to yield a yellow oil (1.09 g, 57.7%).

Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP427) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (150 mg) and (R)-tetrahydrofuran-3-yl methanesulfonate (75 mg, 0.45 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 422.1, found 421.9.

O O

OH OMs

425

O O

OH OMs

426

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OHNH2

N

N NN

Cl

OBnNH2

427 428

356 O O

115

Synthesis of 5-(4-amino-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP428) 3-(3-(benzyloxy)-4-chlorophenyl)-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.4 mg, 11.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 332.1, found 331.9.

Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP429) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (150 mg) and (S)-tetrahydrofuran-3-yl methanesulfonate (75 mg, 0.45 mmol) were added and the reaction was heated to 80º C for overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 422.1, found 421.9. Synthesis of 5-(4-amino-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP430) 3-(3-(benzyloxy)-4-chlorophenyl)-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.9 mg, 12.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 332.1, found 331.9.

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OHNH2

N

N NN

Cl

OBnNH2

429 430

356 O O

116

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP431) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and (R)-tetrahydrofuran-3-yl methanesulfonate (100 mg, 0.6 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 406.2, found 406.0. Synthesis of 5-(4-amino-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP432) 3-(3-(benzyloxy)-4-fluorophenyl)-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (5.7 mg, 12.1% yield over two steps). ESI-MS (M+H)+ m/z calcd 316.1, found 316.0.

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP433) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and (S)-tetrahydrofuran-3-yl methanesulfonate (100 mg, 0.6 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

431 432

367 O O

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

433 434

367 O O

117

three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 406.2, found 406.0. Synthesis of 5-(4-amino-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP434) 3-(3-(benzyloxy)-4-fluorophenyl)-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (7.0 mg, 15% yield over two steps). ESI-MS (M+H)+ m/z calcd 316.1, found 316.0.

Synthesis of tert-butyl (S)-2-yl-propylcarbamate methanesulfonate (ZK437) tert-butyl (S)-2-hydroxypropylcarbamate (10 g, 57 mmol) was dissolved in CH2Cl2 (100 mL) and triethylamine (24 mL, 171 mmol). The reaction was cooled on ice and methanesulfonyl chloride (8.8 mL, 114 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted three times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (20% EtOAc/Hexanes) to yield an oily solid (9.9 g, 68%).

Synthesis of tert-butyl (R)-2-yl-propylcarbamate methanesulfonate (ZK438) tert-butyl (R)-2-hydroxypropylcarbamate (15.0 g, 85.8 mmol) was dissolved in CH2Cl2 (200 mL) and triethylamine (35.5 mL, 257 mmol). The reaction was cooled on ice and methanesulfonyl chloride (13.2 mL, 171 mmol) was carefully added dropwise. The reaction was allowed to proceed overnight at RT, then added to water and extracted four times with CH2Cl2. The combined organic phases were concentrated in vacuo and purified by silica gel chromatography (20% EtOAc/Hexanes) to yield an yellow oil (12.38 g, 57%).

HN

OH

O

O HN

OMs

O

O

437

HN

OH

O

O HN

OMs

O

O

438

118

Synthesis of tert-butyl (R)-2-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate (PP449) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (250 mg) and tert-butyl (S)-2-yl-propylcarbamate methanesulfonate (151 mg, 0.6 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 493.2, found 493.0. Synthesis of 5-(4-amino-1-((R)-1-aminopropan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP450) tert-butyl (R)-2-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (12.4 mg, 27.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 303.1, found 303.0.

Synthesis of tert-butyl (R)-2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate (PP451) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (250 mg) and tert-butyl (S)-2-yl-propylcarbamate methanesulfonate (151 mg, 0.6 mmol) were added and the reaction was heated to 80º C for 1 h. Water was then added and the reaction as

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

449 450

367 HN NH2

O

O

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OHNH2

N

N NN

Cl

OBnNH2

451 452

356 HN NH2

O

O

119

extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 509.2, found 509.0. Synthesis of 5-(4-amino-1-((R)-1-aminopropan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP452) tert-butyl (R)-2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (28 mg, 62% yield over two steps). ESI-MS (M+H)+ m/z calcd 319.1, found 319.0.

Synthesis of tert-butyl (S)-2-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate (PP453) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (250 mg) and tert-butyl (S)-2-yl-propylcarbamate methanesulfonate (151 mg, 0.6 mmol) were added and the reaction was heated to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 493.2, found 493.1. Synthesis of 5-(4-amino-1-((S)-1-aminopropan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP454) tert-butyl (S)-2-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (2.0 mg, 4.5% yield over two steps). ESI-MS (M+H)+ m/z calcd 303.1, found 303.1.

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

453 454

367 HN NH2

O

O

120

Synthesis of tert-butyl (S)-2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate (PP455) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (250 mg) and tert-butyl (S)-2-yl-propylcarbamate methanesulfonate (151 mg, 0.6 mmol) were added and the reaction was heated to 80º C for 1 h. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 509.2, found 509.1. Synthesis of 5-(4-amino-1-((S)-1-aminopropan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP456) tert-butyl (S)-2-(4-amino-3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propylcarbamate was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (6.5 mg, 14% yield over two steps). ESI-MS (M+H)+ m/z calcd 319.1, found 319.0.

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP460) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and bromocyclobutane (0.071 mL, 0.75 mmol) were added and the reaction was heated to 80º C overnight. Water was then added and the reaction as extracted three times with

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OHNH2

N

N NN

Cl

OBnNH2

455 456

356 HN NH2

O

O

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

460 461

367

121

CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 390.2, found 390.1. Synthesis of 5-(4-amino-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP461) 3-(3-(benzyloxy)-4-fluorophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (5 mL). Concentrated HCl (0.5 mL) was added and the reaction was heated to reflux overnight. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (12.3 mg, 27.6% yield over two steps). ESI-MS (M+H)+ m/z calcd 300.1, found 300.1.

Synthesis of 3-(3-(benzyloxy)-4-chlorophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP462) 3-(3-(benzyloxy)-4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and bromocyclobutane (0.075 mL, 0.75 mmol) were added and the reaction was heated to 80º C for 2 hours. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 406.1, found 406.0. Synthesis of 5-(4-amino-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-chlorophenol (PP463) 3-(3-(benzyloxy)-4-chlorophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (5 mL). Concentrated HCl (0.5 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (15.2 mg, 33.9% yield over two steps). ESI-MS (M+H)+ m/z calcd 316.1, found 316.0.

N

N NH

N

Cl

OBnNH2

N

N NN

Cl

OHNH2

N

N NN

Cl

OBnNH2

462 463356

122

Synthesis of 3-(3-(benzyloxy)-4-fluorophenyl)-1-cycloheptyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP464) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and bromocycloheptane (0.10 mL, 0.75 mmol) were added and the reaction was heated to 80º C for 2 hours. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 432.2, found 432.1. Synthesis of 5-(4-amino-1-cycloheptyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-fluorophenol (PP465) 3-(3-(benzyloxy)-4-fluorophenyl)-1-cycloheptyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (5 mL). Concentrated HCl (0.5 mL) was added and the reaction was heated to reflux for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (13.6 mg, 26.7% yield over two steps). ESI-MS (M+H)+ m/z calcd 342.2, found 342.1.

Synthesis of 4-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butanenitrile (PP468) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and 4-bromobutanenitrile (0.075 mL, 0.75 mmol) were added and the reaction was heated to

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

464 465

367

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

468 469

367CN CN

123

80º C for 20 minutes. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 403.2, found 403.0. Synthesis of 4-(4-amino-3-(3-hydroxy-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butanenitrile (PP469) 4-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)butanenitrile was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to 65º C for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (11.8 mg, 25.3% yield over two steps). ESI-MS (M+H)+ m/z calcd 313.1, found 313.0.

Synthesis of 5-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanenitrile (PP470) 3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.15 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and 4-bromopentanenitrile (0.087 mL, 0.75 mmol) were added and the reaction was heated to 80º C for 20 minutes. Water was then added and the reaction as extracted three times with CH2Cl2. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 417.2, found 417.0. Synthesis of 5-(4-amino-3-(3-hydroxy-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanenitrile (PP471) 5-(4-amino-3-(3-(benzyloxy)-4-fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pentanenitrile was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to 65º C for 2 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a off-white solid (7.8 mg, 16% yield over two steps). ESI-MS (M+H)+ m/z calcd 327.1, found 327.0.

N

N NH

N

F

OBnNH2

N

N NN

F

OHNH2

N

N NN

F

OBnNH2

470 471

367 CN CN

124

Synthesis of 3-(benzyloxy)-4-bromobenzoyl chloride (ZK472) 3-(benzyloxy)-4-bromobenzoic acid (5.0 g, 16 mmol) was added to CH2Cl2 (100 mL). Anhydrous DMF (20 drops) was added and the solution was cooled on ice. Oxalyl chloride (14.1 mL, 163 mmol) was added dropwise, and the reaction was then allowed to warm to RT. The reaction was stirred at RT overnight, concentrated in vacuo, and then reconcentrated twice from CH2Cl2. The solid was placed on a high-vacuum line overnight, and then taken onto the next step without further characterization. Synthesis of 2-((3-(benzyloxy)-4-bromophenyl)(hydroxy)methylene)malononitrile (ZK473) NaH (2.6 g, 60 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (1.6 g, 25 mmol) in THF (50 mL) on ice. 3-(benzyloxy)-4-bromobenzoyl chloride (16 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 1 h and then allowed to proceed at RT for 3 hours. 1N HCl (100 mL) was added and the solution was extracted four times with EtOAc (200 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to yield a red solid that was taken onto the next step without further characterization. Synthesis of 2-((3-(benzyloxy)-4-bromophenyl)(methoxy)methylene)malononitrile (ZK474) 2-((3-(benzyloxy)-4-bromophenyl)(hydroxy)methylene)malononitrile (16 mmol) and sodium bicarbonate (9.6 g, 114 mmol) were combined in a solution of 1,4-dioxane (50 mL) and water (6 mL). Dimethylsulphate (12.5 mL, 130 mmol) was slowly added, the reaction was heated to 80º C for 1 h, and then cooled to RT overnight. Water was added, and the aqueous phase was then extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried with MgSO4, filtered, and concentrated in vacuo. The resultant oil was purified by silica gel chromatography (10-20%

BrOBn

OHO

BrOBn

OCl

BrOBn

ONC

CN

BrOBn

OMeNC

CN

Br

OBn

NH

NNC

H2N

N

N NH

N

Br

OBnNH2

472 473 474

475476

125

EtOAc/Hexanes) to yield an orange oil (5.54 g, 92.1% over three steps) Synthesis of 5-amino-3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazole-4-carbonitrile (ZK475) 2-((3-(benzyloxy)-4-bromophenyl)(methoxy)methylene)malononitrile (5.54 g, 15 mmol) and hydrazine monohydrate (0.825 mL, 17 mmol) were combined in ethanol (20 mL) and heated to 80º C for 30 minutes. The reaction was then concentrated in vacuo to yield a white solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 369.0, found 368.9. Synthesis of 3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP476) 5-amino-3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazole-4-carbonitrile was dissolved in formamide (20 mL) and heated to 160º C overnight. The next day the reaction was cooled to RT and a thick precipitate formed. The precipitate was washed with water (50 mL) and purified by two consecutive silica gel chromatographies (2% MeOH in CH2Cl2, followed by 5% MeOH in CH2Cl2) to yield an off-white solid (410 mg, 6.9% yield over two steps). ESI-MS (M+H)+ m/z calcd 396.0, found 395.9.

Synthesis of 2-(hydroxy(3,4-dimethoxyphenyl)methylene)malononitrile (ZK480) NaH (8 g, 200 mmol, 60% dispersion in paraffin oil) was added to a solution of malononitrile (5 g, 75 mmol) in THF (50 mL) on ice. 3,4-dimethoxybenzoyl chloride (10 g, 50 mmol) was dissolved in THF (50 mL) and added the first solution dropwise by syringe at 0º C. The reaction was stirred on ice for 1 h and then allowed to proceed at RT for 3 hours. 1N HCl (100 mL) was added and the solution was extracted three times with EtOAc (200 mL). The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to yield a red solid that was taken onto the next step without further characterization.

OMeOMe

OCl

OMeOMe

ONC

CN

OMeOMe

OMeNC

CN

OMe

OMe

NH

NNC

H2N

N

N NH

N

OMe

OMeNH2

480 481 482

483

126

Synthesis of 2-(methoxy(3,4-dimethoxyphenyl)methylene)malononitrile (ZK481) 2-(hydroxy(3,4-dimethoxyphenyl)methylene)malononitrile (50 mmol) and sodium bicarbonate (29.4 g, 350 mmol) were combined in a solution of 1,4-dioxane (150 mL) and water (18 mL). Dimethylsulphate (38 mL, 400 mmol) was slowly added, the reaction was heated to 80º C for 1 h, and then cooled to RT overnight. Water was added, and the aqueous phase was then extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried with MgSO4, filtered, and concentrated in vacuo. The resultant oil was purified by silica gel chromatography (20-50% EtOAc/Hexanes) to yield an off-white solid (5.4 g, 44% over two steps). ESI-MS (M+H)+ m/z calcd 245.1, found 245.0. Synthesis of 5-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile (ZK482) 2-(methoxy(3,4-dimethoxyphenyl)methylene)malononitrile (4.00 g, 16.4 mmol) and hydrazine monohydrate (1.80 mL, 36 mmol) were combined in ethanol (20 mL) and heated to 80º C for 1 hour. The reaction was then concentrated in vacuo to yield a white solid that was taken onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 245.1, found 245.0 Synthesis of 3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP483) 5-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile (16 mmol) was dissolved in formamide (25 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT and a thick precipitate formed. The precipitate was washed with water and purified by silica gel chromatography (10% MeOH in CHCl3) to yield an orange solid (757 mg, 17.0% yield over two steps). ESI-MS (M+H)+ m/z calcd 272.1, found 272.1.

Synthesis of 1-tert-butyl-5-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile (ZK484) 2-(methoxy(3,4-dimethoxyphenyl)methylene)malononitrile (1.4 g, 5.7 mmol) and tert-butyl hydrazine hydrochloride (852 mg, 6.84 mmol) were combined in ethanol (10 mL) and heated to 80º C for 1 hour. The reaction was then concentrated in vacuo, water was added and the aqueous phase extracted three times with diethyl ether. The organic extract was dried with MgSO4 and concentrated in vacuo to yield a yellow solid (1.7 g, 99%). ESI-MS (M+H)+ m/z calcd 301.2, found 301.0. Synthesis of 1-tert-butyl-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP485) 1-tert-butyl-5-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile (1.7 g, 5.7 mmol) was dissolved in formamide (10 mL) and heated to 180º C overnight. The next day the reaction was cooled to RT, and the solid was washed with water and

OMeOMe

OMeNC

CN

OMe

OMe

NN

NC

H2NN

N NN

OMe

OMeNH2

481 484 485

127

purified by silica gel chromatography (1-2% MeOH in CHCl3) to yield a tan solid (675 mg, 36.4%). 1H NMR (CDCl3) δ 8.35 (s, 1H), 7.24–7.22 (m, 2H), 7.01 (d, J=7.8 Hz, 1H), 5.60 (br, 2H), 3.96 (s, 1H), 3.95 (s, 1H), 1.84 (s, 9H). ESI-MS (M+H)+ m/z calcd 328.2, found 328.1.

Synthesis of 3-(3-(benzyloxy)-4-bromophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (ZK486) 3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.076 mmol) was dissolved in DMF (2 mL). Cesium carbonate (200 mg) and isopropyl iodide (0.025 mL, 0.25 mmol) were added and the reaction was allowed to proceed at RT for 2 hours. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 438.1, found 437.9. Synthesis of 5-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-bromophenol (PP487) 3-(3-(benzyloxy)-4-bromophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to 80º C for 1 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (21.3 mg, 80.8% yield over two steps). ESI-MS (M+H)+ m/z calcd 348.0, found 347.9. ESI-HRMS (M+H)+ m/z calcd 348.0460, found 348.0448.

Synthesis of 3-(3-(benzyloxy)-4-bromophenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP488)

N

N NH

N

Br

OBnNH2

476

N

N NN

Br

OBnNH2

486

N

N NN

Br

OHNH2

487

N

N NH

N

Br

OBnNH2

476

N

N NN

Br

OBnNH2

488

N

N NN

Br

OHNH2

489

MeMe

128

3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.076 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and methyl iodide (0.008 mL, 0.13 mmol) were added and the reaction was allowed to proceed at RT for 90 minutes. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo, and the product carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 410.1, found 409.9. Synthesis of 5-(4-amino-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-bromophenol (PP489) 3-(3-(benzyloxy)-4-bromophenyl)-1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to 80º C for 1 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (25.6 mg, quant. over two steps). ESI-MS (M+H)+ m/z calcd 320.0, found 319.9.

Synthesis of 3-(3-(benzyloxy)-4-bromophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP490) 3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.076 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and cyclobutyl bromide (0.042 mL, 0.45 mmol) were added and the reaction was heated to 80º C for 1 h. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo to yield a solid which was carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 450.1, found 449.9. Synthesis of 5-(4-amino-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-bromophenol (PP491) 3-(3-(benzyloxy)-4-bromophenyl)-1-cyclobutyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (4 mL). Concentrated HCl (0.4 mL) was added and the reaction was heated to 80º C for 1 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (24.6 mg, 90.2% yield over two steps). ESI-MS (M+H)+ m/z calcd 360.0, found 359.9.

N

N NH

N

Br

OBnNH2

476

N

N NN

Br

OBnNH2

490

N

N NN

Br

OHNH2

491

129

Synthesis of 3-(3-(benzyloxy)-4-bromophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP492) 3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.076 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and cyclopentyl iodide (0.026 mL, 0.22 mmol) were added and the reaction was allowed to proceed at RT for 1 h. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo to yield an orange solid, which was carried onto the next step without further characterization. ESI-MS (M+H)+ m/z calcd 464.1, found 463.9. Synthesis of 5-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-bromophenol (PP493) 3-(3-(benzyloxy)-4-bromophenyl)-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine was dissolved in formic acid (3 mL). Concentrated HCl (0.3 mL) was added and the reaction was heated to 80º C for 1 hours. The reaction was then concentrated in vacuo, redissolved in CH2Cl2 and concentrated in vacuo again. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (35.1 mg, quant. over two steps). ESI-MS (M+H)+ m/z calcd 374.1, found 373.9.

Synthesis of 1-cyclobutyl-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP494) 3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.19 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and cyclobutyl bromide (0.087 mL, 0.92 mmol) were added and the reaction was heated to 80º C for 2 hours. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a

N

N NH

N

Br

OBnNH2

476

N

N NN

Br

OBnNH2

492

N

N NN

Br

OHNH2

493

N

N NH

N

OMe

OMeNH2

483

N

N NN

OMe

OMeNH2

494

130

white solid (32.5 mg, 54.2%). (DMSO–d6) δ 8.38 (s, 1H), 7.24–7.22 (m, 2H), 7.14 (d, J=8.6 Hz, 1H), 5.40–5.31 (m, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 2.75–2.69 (m, 2H), 2.44–2.40 (m, 2H), 1.92–1.87 (m, 2H); 13C NMR (DMSO–d6) δ 155.2, 152.2, 151.3, 149.6, 149.1, 145.2, 124.4, 120.8, 112.3, 111.8, 97.1, 55.6, 55.5, 50.2, 29.3, 14.4. ESI-MS (M+H)+ m/z calcd 326.2, found 326.1. ESI-HRMS (M+H)+ m/z calcd 326.1617, found 326.1608.

Synthesis of (R)-3-(4-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-1-ol (PP495) 3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.19 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and (S)-3-bromo-2-methyl propanol (0.095 mL, 0.92 mmol) were added and the reaction was heated to 80º C for 2 hours. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (43.4 mg, 68.6%) ESI-MS (M+H)+ m/z calcd 344.2, found 344.1.

Synthesis of (S)-3-(4-amino-3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2-methylpropan-1-ol (PP496) 3-(3,4-dimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50 mg, 0.19 mmol) was dissolved in DMF (1 mL). Cesium carbonate (200 mg) and (R)-3-bromo-2-methyl propanol (0.095 mL, 0.92 mmol) were added and the reaction was heated to 80º C for 2 hours. Water was then added and the reaction as extracted three times with CHCl3. The organic phases were combined and concentrated in vacuo. The solid was then dissolved in MeCN/H2O and purified by RP-HPLC (MeCN:H2O:0.1% TFA) to yield a white solid (27.2 mg, 43.0%) ESI-MS (M+H)+ m/z calcd 344.2, found 344.0.

N

N NH

N

OMe

OMeNH2

483

N

N NN

OMe

OMeNH2

495

Me

OH

N

N NH

N

OMe

OMeNH2

483

N

N NN

OMe

OMeNH2

496OH

Me

131

Synthesis of 3-(4-bromo-3-(methoxymethoxy)phenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP502) 3-(3-(benzyloxy)-4-bromophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (280 mg, 0.7 mmol) was dissolved in DMF (15 mL). Cesium carbonate (1 g) and isopropyl iodide (0.21 mL, 2.1 mmol) were added and the reaction was allowed to proceed 2 hours at RT. Water was then added, the aqueous phase extracted three times with CHCl3 and the combined organic extract concentrated in vacuo. The product was then dissolved in formic acid (4 mL) and concentrated HCl (0.4 mL) and the reaction was heated to 80º C for 1 hours. The solvent was then removed in vacuo, the product concentrated three times from CH2Cl2 and then dried overnight under high vacuum. The resultant solid was dissolved in CH2Cl2 (15 mL), chloro(methoxy)methane (0.23 mL, 3.0 mmol) and diisopropylethylamine (1.2 mL, 7.0 mmol) were added, and the reaction was allowed to proceed at RT for 7 hours. A saturated solution of NaHCO3 was added, and the aqueous phase was extracted three times with CHCl3. The organic extracts were combined, concentrated in vacuo, and the product purified by silica gel chromatography (1-4% MeOH/CH2Cl2) to yield a yellow solid (182 mg, 65.7% over three steps). ESI-MS (M+H)+ m/z calcd 392.1, found 391.9.

Synthesis of quinoxalin-6-yl-6-boronic acid (ZK515) 6-bromoquinoxaline (209 mg, 1.00 mmol) and potassium acetate (245 mg) were suspended in THF (12 mL). Pd(OAc)2 (21 mg, 0.031 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (303 mg, 1.2 mmol) and 1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride (26 mg, 0.06 mmol) were added and the reaction was heated to reflux overnight under an argon atmosphere. The reaction was then cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a white solid (44.3 mg, 25.5%). ESI-MS (M+H)+ m/z calcd 175.1, found 175.1.

N

N NH

N

NH2

Br

OBn

N

N NN

NH2

Br

OBn

N

N NN

NH2

Br

OH

N

N NN

NH2

Br

O O

476 486 487 502

NN

NN

515

Br B(OH)2

132

Synthesis of 2-methylquinolin-6-yl-6-boronic acid (ZK505) 6-bromo-2-methylquinoline (1.0 g, 5.3 mmol) and triisopropyl borate (1.5 mL, 6.5 mmol) were dissolved in a solution of toluene (8 mL) and THF (2 mL). The reaction was cooled to -80º C under an argon atmosphere, and n-butyllithium (2.6 mL of 2.5 M solution in hexanes, 6.5 mmol) was added over 1 h by syringe pump. The reaction was then warmed to 0º C and quenched with 2N HCl (5 mL). The organic and aqueous phases were separated and allowed to stand overnight at RT. The following day, product had crystallized from the aqueous phase. The crystals were collected, dissolved in MeCN/H2O, and purified by RP-HPLC (MeCN/H2O) to yield a white solid. ESI-MS (M+H)+ m/z calcd 188.1, found 188.1

Synthesis of isoquinolin-6-yl-6-boronic acid (PP127) 6-bromoisoquinoline (500 mg, 2.4 mmol) and triisopropyl borate (0.67 mL, 2.9 mmol) were dissolved in a solution of toluene (4 mL) and THF (1 mL). The reaction was cooled to -80º C under an argon atmosphere, and n-butyllithium (1.2 mL of 2.5 M solution, 2.9 mmol) was added over 1 h by syringe pump. The reaction was then warmed to 0º C and quenched with 2N HCl (2 mL). The organic and aqueous phases were separated and allowed to stand overnight at RT. The following day, product had crystallized from the aqueous phase. The crystals were collected, dissolved in MeCN/H2O, and purified by RP-HPLC (MeCN/H2O) to yield a white solid (32.5 mg, 7.8%). ESI-MS (M+H)+ m/z calcd 174.1, found 174.1.

Synthesis of isoquinolin-7-yl-7-boronic acid (ZK528) 7-bromoisoquinoline (100 mg, 0.48 mmol) and triisopropyl borate (0.14 mL, 0.63 mmol) were dissolved in a solution of toluene (2 mL) and THF (2 mL). The reaction was cooled to -80º C under an argon atmosphere, and n-butyllithium (0.25 mL of 2.5 M

N N

127

Br B(OH)2

N N

528

Br B(OH)2

N N

505

Br B(OH)2

Me Me

133

solution, 0.63 mmol) was added over 30 minutes by syringe pump. The reaction was allowed to proceed at -80º C for an additional 2 hours, then warmed to 0º C and stirred for an additional 30 minutes. The reaction was then quenched by the addition of water (5 mL) and additional toluene was added (5 mL). The organic and aqueous phases were separated and the aqueous phase was lyophilized to yield a white solid (70.8 mg, 85%). ESI-MS (M+H)+ m/z calcd 174.1, found 174.1

Synthesis of 2-chloroquinolin-6-yl-6-boronic acid (ZK526) 6-bromo-2-chloroquinoline (500 mg, 2.1 mmol) and triisopropyl borate (0.6 mL, 2.6 mmol) were dissolved in a solution of toluene (8 mL) and THF (7 mL). The reaction was cooled to -80º C under an argon atmosphere, and n-butyllithium (1.03 mL of 2.5 M solution, 2.6 mmol) was added over 1 h by syringe pump. The reaction was warmed to 0º C, allowed to proceed an additional 1 h. The reaction was then quenched by the addition of water (10 mL) and additional toluene was added (15 mL). The organic and aqueous phases were separated and the product was isolated from the aqueous phase by RP-HPLC (MeCN/H2O) to yield a white solid (450 mg, quant.). ESI-MS (M+H)+ m/z calcd 208.0, found 208.0.

Synthesis of 2-methylquinolin-7-yl-7-boronic acid (ZK531) 7-chloro-2-methylquinoline (500 mg, 2.82 mmol) and potassium acetate (686 mg) were suspended in THF (15 mL). Pd(OAc)2 (57 mg, 0.085 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (860 mg, 3.4 mmol) and 1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride (72 mg, 0.17 mmol) were added and the reaction was heated to reflux overnight under an argon atmosphere. The reaction was then cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a white solid (91.6 mg, 17.3%). ESI-MS (M+H)+ m/z calcd 188.1, found 188.1.

N N

Cl Cl

B(OH)2Br

526

N NN

N NN

533

Br B(OH)2

N N

531Cl B(OH)2

Me Me

134

Synthesis of pyrido[3,2-b]pyrazin-7-yl-7-boronic acid (ZK533) 7-bromopyrido[3,2-b]pyrazine (419 mg, 2.00 mmol) and potassium acetate (490 mg, 5.0 mmol) were suspended in THF (10 mL). Pd(OAc)2 (40 mg, 0.06 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (607 mg, 2.4 mmol) and 1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride (51 mg, 0.12 mmol) were added and the reaction was heated to reflux overnight under an argon atmosphere. The reaction was then cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a grey solid (15 mg, 4.3%). ESI-MS (M+H)+ m/z calcd 176.1, found 176.4.

Synthesis of 2-hydroxyquinolin-6-yl-6-boronic acid (ZK535) 6-chloroquinolin-2-ol (1.0 g, 5.6 mmol) and potassium acetate (1.38 g, 14 mmol) were suspended in THF (20 mL). Pd(OAc)2 (114 mg, 0.17 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.7 g, 6.7 mmol) and 1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride (144 mg, 0.34 mmol) were added and the reaction was heated to reflux overnight under an argon atmosphere. The reaction was then cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a white solid (97 mg, 9.2%). ESI-MS (M+H)+ m/z calcd 190.1, found 190.4.

Synthesis of tert-butyl 6-chloroquinolin-4-ylcarbamate (ZK536) Di-tert-butyl dicarbonate (1.54 g, 7.06 mmol) was dissolved in THF (10 mL) and 6-chloroquinolin-4-amine (700 mg, 3.9 mmol) was added in portions at RT. The reaction was allowed to proceed at RT for 3 days, and then concentrated in vacuo and purified by RP-HPLC (MeCN/H2O) to yield a white solid (916 mg, 83.8%). ESI-MS (M+H)+ m/z calcd 279.1, found 279.4. Synthesis of tert-butyl quinolin-4-ylcarbamate 6-boronic acid (ZK538) tert-butyl 6-chloroquinolin-4-ylcarbamate (600 mg, 2.12 mmol) and potassium acetate (519 mg, 5.3 mmol) were suspended in THF (10 mL). Pd(OAc)2 (43 mg, 0.064

N

Cl

OH

N

B(OH)2

OH

535

N

Cl

NH2

N

Cl

NH

O

O N

B(OH)2

NH

O

O

536 538

135

mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (645 mg, 2.55 mmol) and 1,3-Bis(2,6-diisopropylphenyl)imidazolium chloride (55 mg, 0.14 mmol) were added and the reaction was heated to reflux overnight under an argon atmosphere. The reaction was then cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a white solid (95 mg, 15%). ESI-MS (M+H)+ m/z calcd 289.1, found 289.4.

Synthesis of 1,4-dihydro-4-oxoquinazolin-6-yl-6-boronic acid (ZK532) Bis(dibenzylideneacetone)palladium (17 mg, 0.03 mmol) and tricyclohexylphosphine (20 mg, 0.072 mmol) were dissolved in dioxane (6 mL) and stirred at RT under argon for 30 minutes. 6-iodoquinazolin-4(1H)-one (250 mg, 0.92 mmol), potassium acetate (147 mg, 1.5 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (303 mg, 1.2 mmol) were added and the reaction was heated to 80º C overnight. The following day the reaction was cooled to RT, filtered through a silica plug, and concentrated in vacuo. The product was dissolved in MeCN/H2O and purified by RP-HPLC (MeCN/H2O) to yield a white solid (10.5 mg, 6.0%). ESI-MS (M+H)+ m/z calcd 191.1, found 191.3.

NHN

O

I

NHN

O

B(OH)2

532

Targeted polypharmacology: Discovery of dual inhibitors of ......1 Targeted polypharmacology: Discovery of dual inhibitors of tyrosine and phosphoinositide kinases Beth Apsel1, Jimmy

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