Development of highly potent and selective diaminothiazole inhibitors of. cyclin-dependent kinases

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1 Supporting Information Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases Ernst Schonbrunn a*, Stephane Betzi a, Riazul Alam a, Mathew P. Martin a, Andreas Becker a, Huijong Han a, Rawle Francis b, Ramappa Chakrasali b, Sudhakar Jakkaraj b, Aslamuzzaman Kazi a, Said M. Sebti a, Christopher L. Cubitt c, Anthony W. Gebhard d, Lori A. Hazlehurst d, Joseph S. Tash e, and Gunda I. Georg b* a Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Magnolia Drive, Tampa, FL b Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, University of Minnesota, 717 Delaware Street SE, Minneapolis, MN c Translational Research Laboratory, H. Lee Moffitt Cancer Center and Research Institute, Magnolia Drive, Tampa, FL d Molecular Oncology Department, H. Lee Moffitt Cancer Center and Research Institute, Magnolia Drive, Tampa, FL e Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS S1

2 Table of Contents Title..S1 Table of contents...s2 Summary of data collection and structure refinement...s3 Previously reported aminothiazole inhibitors......s4 Inhibitory activity of analogues against CDK2-cyclin A S6 Profiling of compound 51 against a panel of 339 kinases (Reaction Biology Corp)..S7 Dose-response curves using the DiscoveRx fluorescence-based assay.....s17 Dose-response curves using the P33-radiolabeled assay...s18 Profiling against 48 kinases (Caliper)....S19 Inhibition of different CDK-cyclin complexes and GSK3β by compound 51...S20 Fo-Fc electron density omit maps of representative CDK2-inhibitor complexes...s21 Anti-proliferation dose-response curves against MDA-MB-468 cells..s22 Representative compound NMR spectra....s23 S2

3 Table S1: Summary of data collection and refinement for CDK2-inhibitor complexes determined during the SAR studies. Values in parentheses refer to the highest resolution shell. S3

4 Table S2: Previously reported aminothiazole inhibitors of CDK5 and CHK1. Compound Modifications PDB Reported Inhibitory values R 2 O R 1 N S N H Cl R 1 : -NH 2 R 2 : 3-NO 2 3O0G R 1 : -NH 2 R 2 : 2,4-di-Cl - CDK5: 0.7 μm R 1 : -NH 2 R 2 : 3-CF 3 - CDK5: 38 μm CDK5/p25: 2.0μM, GSK3β: 0.7μM, CDK1: 4.0μM, CK1: 124.8μM, PKA: 250.3μM, PKC: >1000μM, Src: >1000μM. R 1 : -NH 2 R 2 : 4-Cl - CDK5: 66 μm R 1 : -H R 2 : 2,4-di-Cl - CDK5: >100 μm - 2WMS CHK1: 36.9 (+/- 1.7) % at 250 μm R 1 : 4-ClPh R 2 : 3-NO 2 Ph R 3 : -H 3O0G CDK5: 0.75μM R 1 : 4-ClPh R 2 : 3-FPh R 3 : -H - CDK5: 0.7μM R 1 : 4-ClPh R 2 : 3-OMePh R 3 : -H - CDK5: 4.0μM R 1 : 3-Piperidyl R 2 : 3-FPh R 3 : -H - CDK5: 0.5μM R 1 : 4-Piperidyl R 2 : 3-FPh R 3 : -H - CDK5: 6.2μM R 1 : 3-NH 2 Ph R 2 : 3-FPh R 3 : -H - CDK5: 0.031μM R 1 : 3-NHMePh R 2 : 3-FPh R 3 : -H - CDK5: 0.044μM R 1 : 4-NMe 2 Ph R 2 : 3-FPh R 3 : -H - CDK5: 0.068μM R 1 : 2-Py R 2 : 3-FPh R 3 : -H - CDK5: 0.033μM R 1 : 2-Py R 2 : 2-FPh R 3 : -H - CDK5: 0.038μM R 1 : 2-Py R 2 : 4-FPh R 3 : -H - CDK5: 1.2μM R 1 : 2-Py R 2 : 2-OMePh R 3 : -H - CDK5: 0.080μM R 1 : 2-Py R 2 : 3-OMePh R 3 : -H - CDK5: 0.40μM R 1 : 2-Py R 2 : 4-OMePh R 3 : -H - CDK5: 1.1μM R 1 : 3-Py R 2 : 2-FPh R 3 : -H - CDK5: 0.030μM S4

5 R 1 : 3-Py R 2 : 3-FPh R 3 : -H - CDK5: 0.062μM R 1 : 3-Py R 2 : 4-FPh R 3 : -H - CDK5: 4.1μM R 1 : 3-Py R 2 : Ph R 3 : -H - CDK5: 0.34μM R 1 : 3-Py R 2 : 3-NO 2 Ph R 3 : -H - CDK5: 0.13μM R 1 : 3-Py R 2 : 2-OMePh R 3 : -H - CDK5: 0.42μM R 1 : 3-Py R 2 : 3-OMePh R 3 : -H - CDK5: 0.011μM R 1 : 3-Py R 2 : 4-OMePh R 3 : -H - CDK5: 3.5μM R 1 : 4-Py R 2 : 2-FPh R 3 : -H - CDK5: 0.36μM R 1 : 4-Py R 2 : 3-FPh R 3 : -H - CDK5: 0.018μM R 1 : 4-Py R 2 : 4-FPh R 3 : -H - CDK5: 0.40μM R 1 : 4-Py R 2 : 2-OMePh R 3 : -H - CDK5: 0.06μM R 1 : 4-Py R 2 : 3-OMePh R 3 : -H - CDK5: 0.20μM R 1 : 4-Py R 2 : 4-OMePh R 3 : -H - CDK5: 0.60μM R 1 : 3-Py R 2 : 2-Py R 3 : -H - CDK5: 0.80μM R 1 : 3-Py R 2 : 3-Py R 3 : -H - CDK5: 0.10μM R 1 : 3-Py R 2 : 4-Py R 3 : -H - CDK5: 1.71μM R 1 : 3-Py R 2 : 2-FPh R 3 : -Me - CDK5: >10.0μM R 1 : 3-(6-CF 3 )Py R 2 : 2-FPh R 3 : -H - CDK5: 0.16μM S5

6 Tables S3-S6: CDK2-cyclin A2 inhibitory activities of analogues of hit compound 1. Values represent the results for duplicate or triplicate dose-response measurements with a standard deviation of ± 10 %. Table S3 R O Compound IC 50 (µm) PDB ID 3R9N - 3RAH Table S4 HOOC R1 SO 2 NH R2 O 2 N N N N O 2 N Compound IC 50 (µm) >100 >100 PDB ID S6

7 Table S5 NH 2 F R Compound IC 50 (µm) PDB ID RNI - S7

8 Table S6 N R NH NH N NH Compound IC 50 (µm) >100 >100 >100 PDB ID R9D 3R9O - 3R9H R O C O NH Compound IC 50 (µm) >100 >100 >100 >100 >100 >100 PDB ID CDK1/2 inhibitor III F NH F S N N H 2 N N N H SO 2 NH 2 Pan-kinase inhibitor IC 50 against CDK2 = < 1 nm (P 33 -labeled assay) PDB ligand code: DKI CAS# S8

9 Table S7: Profiling of compound 51 against a panel of 339 kinases. Residual enzymatic activity (in % of DMSO controls) was determined in single dose duplicate at a compound concentration of 0.1 µm. The ATP concentration was 10 µm. Staurosporine served as a positive control (data not shown). The experiments were performed by Reaction Biology Corp. using a P 33 -radiolabeled assay. KINASE % Activity remaining Data 1 Data 2 CDK5/p CDK5/p CDK2/cyclin A CDK2/cyclin E CDK9/cyclin T CDK2/Cyclin A CDK1/cyclin B CDK9/cyclin K CDK4/cyclin D CDK6/cyclin D CDK1/cyclin E CDK4/cyclin D GSK3a ERK7/MAPK CDK1/cyclin A TAOK2/TAO GSK3b CDK6/cyclin D CLK CLK BMPR DYRK1/DYRK1A SRPK CDK3/cyclin E MUSK CLK TAOK SRPK MLK3/MAP3K KHS/MAP4K DYRK1B IRAK CAMKK SLK/STK CLK TAOK3/JIK IRAK STK S9

10 TYK ZIPK/DAPK MST3/STK GCK/MAP4K WEE RIPK LCK2/ICK CAMKK CAMK1a WNK NLK LOK/STK DMPK CK1g TNIK HGK/MAP4K MEKK WNK BLK CK2a MAPKAPK5/PRAK ZAK/MLTK IKKa/CHUK PKAcg DYRK NEK EPHA MLK2/MAP3K EPHA HIPK TXK MYO3b FGFR PKCb TYK1/LTK PKG1a NEK NEK CAMK HCK Aurora C BTK P38d/MAPK ULK SIK ALK6/BMPR1B MINK/MINK MLCK/MYLK S10

11 STK Aurora B PIM DLK/MAP3K TGFBR STK38/NDR JNK HPK1/MAP4K OSR1/OXSR PLK TRKC NEK ACK BRK FGFR CK1g p70s6kb/rps6kb CDC7/DBF PKCb DYRK CTK/MATK CAMK1d ROCK SGK3/SGKL LATS PAK P38b/MAPK AXL RSK PAK SSTK/TSSK SRMS LCK ARAF BMX/ETK HIPK IKKe/IKBKE YES/YES MLK1/MAP3K GRK CAMK2a PHKg ALK5/TGFBR PDK1/PDPK EPHA PAK BRAF CAMK2d S11

12 ERK5/MAPK FGFR CAMK1b FMS MEK ZAP ABL TIE2/TEK FGFR DYRK GRK CDK7/cyclin H DAPK RSK c-met IRR/INSRR LKB AKT PYK CAMK1g PKCiota ERK2/MAPK ULK PKCnu/PRKD TTBK EPHA c-src TTBK PKCepsilon NEK FLT4/VEGFR MSSK1/STK STK32C/YANK PAK GRK ROCK PKCa TNK MAPKAPK PKCg PDGFRb MNK JNK IGF1R NEK P38a/MAPK ALK2/ACVR MEK S12

13 SIK FER LRRK EPHB SIK CAMK2b TLK JAK CHK JAK PKCmu/PRKD MARK MEKK STK39/STLK NEK EPHA CK1epsilon PLK MKK FLT STK38L/NDR TBK RAF SGK STK22D/TSSK MAPKAPK EPHB TRKB RET PKN1/PRK PLK4/SAK MRCKa/CDC42BPA PLK HIPK PBK/TOPK MYLK PIM CK1a ALK1/ACVRL TLK EGFR FLT1/VEGFR MSK2/RPS6KA RSK TSSK3/STK22C PKCtheta DDR RIPK S13

14 Haspin MEKK KDR/VEGFR DMPK EPHB GRK TRKA IKKb/IKBKB Aurora A c-mer CK2a PKCeta MST1/STK LYN ERBB4/HER DCAMKL CK1g RSK ERBB2/HER MEK ABL2/ARG FAK/PTK MKK PASK DDR MARK c-kit ROS/ROS SYK TEC PKN3/PRK EPHB STK32B/YANK ARK5/NUAK PKG1b CHK HIPK PKAcb LYN B EPHA MST2/STK PKCd ALK3/BMPR1A BRSK COT1/MAP3K GRK MNK P38g S14

15 ALK4/ACVR1B SNARK/NUAK TESK TAK ASK1/MAP3K LIMK PAK NEK MELK JNK PDGFRa VRK MRCKb/CDC42BPB MSK1/RPS6KA CAMK2g VRK MARK AKT ERK PKN2/PRK MLCK2/MYLK NEK SGK LATS WNK TYRO3/SKY PHKg EPHA DCAMKL RON/MST1R IR FES/FPS ALK JAK CSK p70s6k/rps6kb GRK EPHA BRSK DRAK1/STK17A CK1d PIM AKT GRK PKCzeta MST ITK STK25/YSK S15

16 TSSK PKG2/PRKG RIPK PKA FYN DAPK ULK MARK2/PAR-1Ba FGR PAK PRKX PKD2/PRKD FRK/PTK S16

17 Figure S1: Evaluation of inhibitory activity on the activated CDK2-cyclin A2 complex using the DiscoveRx fluorescence-based assay. Shown are dose-response curves for representative compounds. Staurosporine served as a control. IC 50 values and hill coefficients are indicated. S17

18 Figure S2: Inhibitory activities of compounds 42 and 52 against CDK2-cyclin A2 in comparison with staurosporine using the P 33 -radiolabeled assay. S18

19 Figure S3: Broad kinase profiling (Caliper). Representative compounds were tested against 48 kinases and activities are presented as a gradient heat map from green (0% inhibition) to black (50% inhibition) to red (100% inhibition). S19

20 Figure S4: Inhibition of different CDK-cyclin complexes and GSK3β by compound 51. A P 33 -radiolabeled assay was used to determine IC 50 values with staurosporine as a control. See also Table 2 of the main text. The assays were performed by Reaction Biology Corp. S20

Figure S5: Crystallography of representative CDK2-inhibitor complexes.

in which the inhibitor was omitted from the model.

the DFG motif (residues 145-147) in cyan, and other residues in grey.

21 Figure S5: Crystallography of representative CDK2-inhibitor complexes. Shown are Fo-Fc electron density maps contoured at 3σ (red mesh) resulting from refinements in which the inhibitor was omitted from the model. The hinge region (residues 81-84) is shown in orange, the gatekeeper Phe80 residue in red, the DFG motif (residues ) in cyan, and other residues in grey. Protein and compounds (yellow) are shown as ball and stick representations. Black and green dotted lines indicate hydrogen bonding and hydrophobic interactions, respectively. S21

22 Fig S6: Anti-proliferation activity of representative inhibitors against MDA-MB-468 breast cancer cells. IC 50 values were determined in triplicate. S22

23 Representative compound NMR spectra 2 1 H Spectrum S23

24 2 13 C Spectrum S24

25 3 1 H Spectrum S25

26 3 13 C Spectrum S26

27 9 1 H Spectrum S27

28 9 13 C Spectrum S28

29 20 1 H Spectrum S29

30 20 13 C Spectrum S30

31 34 1 H Spectrum S31

32 34 13 C Spectrum S32

33 42 1 H Spectrum S33

34 42 13 C Spectrum S34

35 51 1 H Spectrum O 2 N O H 2 N N S N H SO 2 NH 2 S35

36 51 1 H Spectrum in D 2 O O 2 N O H 2 N N S N H SO 2 NH 2 S36

37 51 13 C Spectrum O 2 N O H 2 N N S N H SO 2 NH 2 S37

38 S38

39 S39

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