pharmacological targeting of the pseudokinase her3€¦ · supplementary information ....

SUPPLEMENTARY INFORMATION

Pharmacological Targeting of the Pseudokinase Her3

Ting Xie1,2,3, Sang Min Lim1,2,3, Kenneth D. Westover4, Michael E. Dodge5, Dalia Ercan5, Scott B. Ficarro2,6, Durga Udayakumar4, Deepak Gurbani4, Hyun Seop Tae7, Steven M. Riddle8, Taebo Sim9,10, Jarrod A. Marto2,6, Pasi A. Jänne5*, Craig M. Crews7*, Nathanael S. Gray1,2* 1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 2Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. 3Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. 4Departments of Biochemistry and Radiation Oncology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA. 5Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston Massachusetts, USA. 6Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 7Department of Molecular, Cellular and Development Biology, Yale University, New Haven, Connecticut, USA. 8Primary and Stem Cell Systems, Life Technologies Corporation, Madison, Wisconsin, USA. 9Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea. 10KU-KIST Graduate School of Converging Science and Technology, Seoul, Republic of Korea. *E-Mail: [email protected], [email protected] or [email protected].

*Correspondence should be addressed to Nathanael S. Gray ([email protected]) or Craig M. Crews ([email protected]) or Pasi A. Jänne ([email protected])

Contents

- Supplementary Results - Supplementary Methods

Supplementary Results:

Nature Chemical Biology: doi:10.1038/nchembio.1658

Supplementary Figure 1

Supplementary Figure 1 | Screening hits and in silico docking study. (a) Chemical structures of top 5 hits based on Lanthascreen® technology (Invitrogen) protein binding assay. (b) Simulated molecular docking suggests that all hit compounds interact with a hydrophobic pocket behind the ATP binding pocket of Her3.



Supplementary Figure 2 | Time dependence of TX1-85-1 IC50s in the LanthaScreenTM binding assay. IC50s were determined at various time points using the LanthaScreenTM binding assay. Each condition was tested in triplicate. Data represent mean values ± s.d. The IC50 decreases over time likely due to covalent binding of the compound. This effect plateaus at about 3 hours.



Supplementary Figure 3 | Mass spectrometry analysis of labeled Her3. (a) Recombinant Her3 protein was incubated with a 10-fold molar excess of N-(5-(4-amino-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-phenoxyphenyl)acrylamide for 3 hours at room temperature and the resulting mixture analyzed by mass spectrometry. (b) MS/MS spectrum of the peptide IPV(C*)IK, where (C*) denotes a compound labeled cysteine residue. Ions of type b and y are shown in blue and red, respectively. Among the four cysteine residues within the Her3 kinase domain, only Cys721, located on the roof of the ATP binding pocket, was covalently modified. (c) The mass spectrometry result of labeling by TX1-85-1 against recombinant Her3 protein.



Supplementary Figure 4 | Addition of a biotin tail to TX1-85-1 doesn’t diminish binding affinity for Her3. The IC50 of TX1-85-1-biotin is 50.7 nM based on Lanthascreen® protein binding assay. Each condition was tested in triplicate. Data represent mean values ± s.d.



Supplementary Figure 5 | RNAi study confirms ErbB3 dependent cell lines. Treatment of PC9 GR4 and Ovcar8 cell lines with shRNA directed against the Her3 transcript shows these lines to be Her3 dependent cell lines while negative controls, A549 and Ovcar5, are not.



Supplementary Figure 6 | EGFR dependent Ba/F3 EGFR VIII cells are sensitive to Erlotinib but 50 fold less sensitive to TX2-121-1. Cells were exposed to compounds for 72 hrs. Each condition was tested in triplicate. Data represent mean values ± s.d. This suggests that anti-proliferative effects of TX2-121-1 on Her3 dependent lines are not from EGFR inhibition.



Supplementary Figure 7 | Sanger DNA sequencing of ErbB3. TGC to TCC missense mutation leads to C721S mutation.



Supplementary Figure 8: TX1-85-1 covalently attaches to Her3 but not Her3 C721S in cell lysates. Her3 pull-down using TX1-85-1-biotin in PC9 GR4 Her3 and PC9 GR4 C721S cell lysate shows a strong interaction between TX1-85-1-biotin and Her3 WT, but not Her3 C721S. Her3 C721S lacks the capacity to covalently bind to the acryloyl-amide motif of TX1-85-1-biotin suggesting that the observed interaction between WT Her3 and TX1-85-1-biotin is dependent on a covalent interaction.



Supplementary Figure 9 | EC50 measurements for PC9 GR4 cells exposed to TX1-85-1 and other derivatives. PC9 GR4 Her3 WT and PC9 GR4 Her3 C721S cells were exposed to TX1-85-1, TX1-85-3, TX2-121-1 and TX2-135-2 at the indicated concentrations and cell viability measured at 72 hrs using the CellTiter-Glo® assay. Each condition was tested in triplicate. Data represent mean values ± s.d.



Supplementary Figure 10 | Her3 signaling axis is inhibited by TX1-85-1 and TX2-121-1 in HCC2935 cells. 80% confluent cells were treated with Her3 inhibitors with indicated concentration for 12 hours. The cells were then stimulated with 50 ng/ml HRG for 20 min and then harvested for western blot analysis. Treatment with both compounds reduce levels of total Her3 and phosphorylated Her3. Reductions in total Akt and phospho Akt and Erk levels are also observed.



Supplementary Figure 11 | Her3 expression in Her3-dependent and independent cell lines. Total cell lysates for the 7 cell lines used in in this study were separated by SDS PAGE and blotted for total Her3 levels. Her3 dependent cell lines, based on prior studies included PC9 GR4, HCC2935, Ovcar 8, PC9 GR4 Her3 WT and PC9 GR4 C721S. Her3 independent cell lines included A549 and Ovcar 5. Tubulin was used as a loading control. For this set of lines, Her3-dependent lines express Her3 protein at higher levels than Her3-independent lines.


Supplementary Table 1: KiNativ® profiling of TX1-85-1 and TX2-121-1. KiNativ® profiling data shows that TX2-121-1 is a relatively selective binder of Her3 but also binds to Her2, Lyn and several other Src family kinases. Itis similar to TX1-85-1, but Her3 inhibition score is higher than other kinases.

Kinase Sequence

TX2-121-1 (2 μM)

TX1-85-1

(2 μM)

HER3/ErbB3 GVWIPEGESIKIPVCIKVIEDK 90.3 75.9

YES VAIKTLKPGTMMPEAFLQEAQIMK 84.9 ND

LYN VAVKTLKPGTMSVQAFLEEANLMK 77.8 79.8

SRC VAIKTLKPGTMSPEAFLQEAQVMKK 70.8 ND

FYN, SRC, YES QGAKFPIKWTAPEAALYGR 70.5 65.7

EGFR IPVAIKELR 65.8 43.6

HER2/ErbB2 GIWIPDGENVKIPVAIKVLR 62.6 69.8

MSK2 domain1 DLKLENVLLDSEGHIVLTDFGLSK 60.4 ND

TEC YVLDDQYTSSSGAKFPVK 59.2 ND

CSK VSDFGLTKEASSTQDTGKLPVK 53.2 34.4

EGFR LLGAEEKEYHAEGGKVPIK 48.3 24.9

MAP2K2 HQIMHRDVKPSNILVNSR 40 ND

FRK HEIKLPVK 38.6 ND

MPSK1 DLKPTNILLGDEGQPVLMDLGSMNQACIHVEGSR 34.1 ND

GCK DIKGANLLLTLQGDVK 33.1 4.7

OSR1 DVKAGNILLGEDGSVQIADFGVSAFLATGGDITR 32.1 ND

MST4, YSK1 DIKAANVLLSEQGDVK 31.8 0.3

AurA FILALKVLFK 30.4 ND

MSK1 domain1 DIKLENILLDSNGHVVLTDFGLSK 30.4 12.9

MAP3K1 DVKGANLLIDSTGQR 30.3 -1.1

PKN2 DLKLDNLLLDTEGFVK 29.5 ND


STLK3 DLKAGNILLGEDGSVQIADFGVSAFLATGGDVTR 29.2 ND

PIK3CB VFGEDSVGVIFKNGDDLRQDMLTLQMLR 28.8 11.6

PHKg1 DLKPENILLDDNMNIK 28.7 ND

PI4K2B SEEPYGQLNPKWTK 28.3 ND

MAP3K4 DIKGANIFLTSSGLIK 27.7 -8.4

STLK5 YSVKVLPWLSPEVLQQNLQGYDAK 27.6 ND

MYO3A, MYO3B DVKGNNILLTTEGGVK 26.9 ND

CCRK DLKPANLLISASGQLK 25.7 ND

KSR1, KSR2 SKNVFYDNGK 25.7 ND

NEK9 DIKTLNIFLTK 25.2 3.2

ZAK WISQDKEVAVKK 24.9 15.7

RSK1 domain1 DLKPENILLDEEGHIKLTDFGLSKEAIDHEK 24.3 ND

SLK DLKAGNILFTLDGDIK 23.7 3.4

PKD2 DVAVKVIDK 23.3 ND

MER, TYRO3 KIYSGDYYR 22.9 ND

MLK1 DLKSSNILILQK 22.9 ND

IRAK4 DIKSANILLDEAFTAK 22.7 7.3

NLK DIKPGNLLVNSNCVLK 22.7 ND

KSR1 SKNVFYDNGKVVITDFGLFGISGVVR 22.6 ND

AurB SHFIVALKVLFK 22.3 ND

LATS2 DIKPDNILIDLDGHIK 22.2 ND

TAK1 DLKPPNLLLVAGGTVLK 22 -4.6

Erk3 DLKPANLFINTEDLVLK 21.7 ND

CLK2 LTHTDLKPENILFVNSDYELTYNLEK 21.6 ND

CHED DIKCSNILLNNR 21.5 ND

PLK2 DLKLGNFFINEAMELK 21.1 ND

MAP3K6 DIKGDNVLINTFSGLLK 20.8 ND


MAP3K5 DIKGDNVLINTYSGVLK 20.6 5.1

GSK3B DIKPQNLLLDPDTAVLK 20.5 -9.3

TAO1, TAO3 DIKAGNILLTEPGQVK 20.3 -7.5

ZC1/HGK, ZC2/TNIK, ZC3/MINK DIKGQNVLLTENAEVK 20.1 -16.4

RSK3 domain1 DLKPENILLDEEGHIKITDFGLSK 19.5 ND

PIK3C3 TEDGGKYPVIFKHGDDLR 19 ND

MST1 ETGQIVAIKQVPVESDLQEIIK 18.9 14

NEK6, NEK7 DIKPANVFITATGVVK 18.5 2

MAP3K2 ELAVKQVQFDPDSPETSKEVNALECEIQLLK 18.3 ND

PI4KB VPHTQAVVLNSKDK 18.3 ND

MAPKAPK3 CALKLLYDSPK 18.2 ND

ACK TVSVAVKCLKPDVLSQPEAMDDFIR 18.1 ND

DNAPK KGGSWIQEINVAEK 18.1 20.6

PKN1 VLLSEFRPSGELFAIKALK 18 ND

VRK2 MLDVLEYIHENEYVHGDIKAANLLLGYK 17.9 13.6

MST2 ESGQVVAIKQVPVESDLQEIIK 17.8 8.2

CHK1 DIKPENLLLDER 17.6 ND

MAP3K2, MAP3K3 DIKGANILR 17.6 0

CDK4 DLKPENILVTSGGTVK 17.4 ND

CLK3 YEIVGNLGEGTFGKVVECLDHAR 17 ND

NEK1 DIKSQNIFLTK 17 -5.4

TLK2 YLNEIKPPIIHYDLKPGNILLVNGTACGEIK 17 ND

TLK1 YLNEIKPPIIHYDLKPGNILLVDGTACGEIK 16.9 9.1

ABL, ARG YSLTVAVKTLKEDTMEVEEFLK 16.7 5.3

CaMK1a LVAIKCIAK 16.7 7.5


GAK DLKVENLLLSNQGTIK 16.5 ND

p38d, p38g DLKPGNLAVNEDCELK 16.5 ND

EphB4 FLEENSSDPTYTSSLGGKIPIR 16.4 ND

Erk5 DLKPSNLLVNENCELK 16.3 7.4

SIK TQVAIKIIDK 16.2 ND

PIP4K2C TLVIKEVSSEDIADMHSNLSNYHQYIVK 16.1 1.4

RSK2 domain1 DLKPENILLDEEGHIKLTDFGLSKESIDHEK 15.9 -4.6

MAP2K6 DVKPSNVLINALGQVK 15.8 -3.2

MARK4 DLKAENLLLDAEANIK 15.6 ND

NEK3 SKNIFLTQNGK 15.6 -4.3

PKCe DLKLDNILLDAEGHCK 15.6 ND

ABL, ARG LMTGDTYTAHAGAKFPIK 15.5 ND

AurA DIKPENLLLGSAGELK 15.5 13.3

AurA, AurB, AurC GKFGNVYLAR 15.5 -1.4

PDK1 EYAIKILEK 15.5 ND

MAP3K3 ELASKQVQFDPDSPETSKEVSALECEIQLLK 15.4 ND

CDC7 DVKPSNFLYNR 14.6 ND

IKKb DLKPENIVLQQGEQR 14.5 -6

GCN2 DLKPVNIFLDSDDHVK 14.3 -2.8

MST3 DIKAANVLLSEHGEVK 14.2 -5.6

CDC2 DLKPQNLLIDDKGTIK 14.1 5.1

JAK1 QLASALSYLEDKDLVHGNVCTKNLLLAR 13.9 11.4

CaMKK1 DIKPSNLLLGDDGHVK 13.8 ND

PIP4K2A AKELPTLKDNDFINEGQK 13.7 -13.3

TAO2 DVKAGNILLSEPGLVK 13.6 1.8

PIK3C2B VIFKCGDDLRQDMLTLQMIR 13.4 ND

CASK ETGQQFAVKIVDVAK 13.3 -2.5


CRK7 DIKCSNILLNNSGQIK 13.3 ND

SGK3 FYAVKVLQK 13.3 -4.4

MAST3 DLKPDNLLITSLGHIK 13.2 -3.6

MAP2K1, MAP2K2 KLIHLEIKPAIR 13.1 ND

PIP5K3 GGKSGAAFYATEDDRFILK 13.1 1.1

GPRK5 DLKPENILLDDYGHIR 13 ND

eEF2K YIKYNSNSGFVR 12.4 1.5

EphA1 LLDDFDGTYETQGGKIPIR 12.4 ND

SRPK1 IIHTDIKPENILLSVNEQYIR 12 -1.7

CaMK2d IPTGQEYAAKIINTKK 11.9 -7.6

LATS1 ALYATKTLR 11.9 2.7

CaMK1d LFAVKCIPK 11.8 -2.5

RIPK1 DLKPENILVDNDFHIK 11.7 ND

p38a DLKPSNLAVNEDCELK 11.3 -10

MASTL LYAVKVVK 11.2 -1.5

CaMKK2 DIKPSNLLVGEDGHIK 10.8 ND

NDR2 DIKPDNLLLDAK 10.8 -7.9

MARK3 EVAIKIIDKTQLNPTSLQK 10.7 -10.9

PCTAIRE1 SKLTDNLVALKEIR 10.7 ND

MAP2K1, MAP2K2 DVKPSNILVNSR 10.6 10.5

CaMK2g TSTQEYAAKIINTK 10.5 -3.2

CHK2 DLKPENVLLSSQEEDCLIK 10.4 ND

MASTL GAFGKVYLGQK 10.4 -5.3

YANK3 DVKPDNILLDER 10.4 -10.8

CDK5 DLKPQNLLINR 10.3 2.1

AKT1 GTFGKVILVK 10.1 ND


LOK DLKAGNVLMTLEGDIR 10 4.2

Wnk1, Wnk2, Wnk3 DLKCDNIFITGPTGSVK 9.7 6.5

NEK8 DLKTQNILLDK 9.5 ND

p38a QELNKTIWEVPER 9.2 16.7

Wnk1, Wnk2 GSFKTVYK 9.2 ND

ULK1 DLKPQNILLSNPAGR 9.1 ND

ILK WQGNDIVVKVLK 9 15.4

MARK3, MARK4 EVAIKIIDK 8.8 ND

TGFbR2 DLKSSNILVK 8.8 ND

MLK3 DLKSNNILLLQPIESDDMEHK 8.7 6.2

MLKL APVAIKVFK 8.7 -4.8

RSK1 domain2 DLKPSNILYVDESGNPECLR 8.7 -9.6

CK1g2 DVKPENFLVGRPGTK 8.6 ND

ZC1/HGK TGQLAAIKVMDVTEDEEEEIKLEINMLKK 8.6 ND

IKKa DLKPENIVLQDVGGK 8.5 -4.5

CDK2 DLKPQNLLINTEGAIK 8.2 2.1

NuaK1 VVAIKSIR 8.2 2.7

PKD3 DVAIKVIDK 8.2 ND

KHS1 NVHTGELAAVKIIK 8.1 -1.2

PKR DLKPSNIFLVDTK 8.1 -1.2

DGKH ATFSFCVSPLLVFVNSKSGDNQGVK 7.6 ND

NEK4 DLKTQNVFLTR 7.6 16.6

PLK1 CFEISDADTKEVFAGKIVPK 7.6 9.5

JNK1, JNK2, JNK3 DLKPSNIVVK 7.4 1.6

DGKA IDPVPNTHPLLVFVNPKSGGK 7.3 ND

GSK3A DIKPQNLLVDPDTAVLK 7.2 -9.9


PRP4 CNILHADIKPDNILVNESK 7 ND

TBK1 TGDLFAIKVFNNISFLRPVDVQMR 7 ND

FER TSVAVKTCKEDLPQELK 6.7 -4.2

PAK2 IGQGASGTVFTATDVALGQEVAIKQINLQK 6.6 ND

PRPK FLSGLELVKQGAEAR 6.3 -14.2

SNRK DLKPENVVFFEK 6.1 ND

STLK6 HTPTGTLVTIKITNLENCNEER 5.9 ND

KHS2 NVNTGELAAIKVIK 5.2 16.4

MAST1, MAST2 DLKPDNLLITSMGHIK 5.2 6.9

FRAP IQSIAPSLQVITSKQRPR 5.1 8.2

IRAK1 AIQFLHQDSPSLIHGDIKSSNVLLDER 5.1 8.5

MAP2K1 IMHRDVKPSNILVNSR 5.1 8.7

JAK1 domain2 IGDFGLTKAIETDKEYYTVK 4.9 7.5

MAP2K7 DVKPSNILLDER 4.9 ND

JAK1 domain2 YDPEGDNTGEQVAVKSLKPESGGNHIADLKK 4.7 13.1

MAP2K4 DIKPSNILLDR 4.7 -0.2

RON IQCAIKSLSR 4.6 ND

NDR1 DIKPDNLLLDSK 4.3 -2.3

SMG1 DTVTIHSVGGTITILPTKTKPK 4.2 -6.8

NuaK2 LVAIKSIR 3.9 ND

DNAPK EHPFLVKGGEDLR 3.7 -2.5

FAK CIGEGQFGDVHQGIYMSPENPALAVAIKTCK 3.6 12.1

CK1g1, CK1g2, CK1g3 KIGCGNFGELR 3.4 ND

NEK2 DLKPANVFLDGK 3.1 ND

RSKL1 VLGVIDKVLLVMDTR 3 ND

Erk2 DLKPSNLLLNTTCDLK 2.8 ND

LKB1 DIKPGNLLLTTGGTLK 2.7 ND


EphB3 FLEDDPSDPTYTSSLGGKIPIR 2.5 ND

ULK3 NISHLDLKPQNILLSSLEKPHLK 2.4 -0.2

Erk1 DLKPSNLLINTTCDLK 2.1 -2.3

MRCKb DIKPDNVLLDVNGHIR 1.8 ND

PCTAIRE2, PCTAIRE3 SKLTENLVALKEIR 1.3 1.2

PITSLRE DLKTSNLLLSHAGILK 1.3 -8.3

MLK4 DLKSSNILLLEK 0.8 1

CDK7 DLKPNNLLLDENGVLK 0 -7

RSK1 domain1, RSK2 domain1, RSK3 domain1 DLKPENILLDEEGHIK 0 7.3

BRAF DLKSNNIFLHEDLTVK -0.1 21.6

RSK2 domain2 DLKPSNILYVDESGNPESIR -0.2 2.6

CLK1 LTHTDLKPENILFVQSDYTEAYNPK -0.3 ND

MAP2K3 DVKPSNVLINK -1.7 2.9

CDK9 DMKAANVLITR -2 -17.7

TNK1 SVPVAVKSLR -2.2 ND

PEK DLKPSNIFFTMDDVVK -2.6 8.3

CK2a2 DVKPHNVMIDHQQK -3.8 ND

IRE1 DLKPHNILISMPNAHGK -4.8 -14.6

PKCa, PKCb DLKLDNVMLDSEGHIK -6.5 ND

MAST4 DLKPDNLLVTSMGHIK -7.4 ND

ANKRD3 TWLAIKCSPSLHVDDR -7.6 ND

CK1a DIKPDNFLMGIGR -7.6 -3.5

p70S6Kb DLKPENIMLSSQGHIK -9.3 -10.3

NEK7 AACLLDGVPVALKK -9.6 -9.5

MET DMYDKEYYSVHNK -10.3 0.9


CK1d, CK1e DVKPDNFLMGLGKK -12.9 ND

Wee1 YIHSMSLVHMDIKPSNIFISR -12.9 3.6

MARK2, MARK3 DLKAENLLLDADMNIK -13.2 -9.5

MELK DLKPENLLFDEYHK -14.2 ND

ROCK1, ROCK2 DVKPDNMLLDK -14.3 -8.4

PYK2 YIEDEDYYKASVTR -15 ND

smMLCK QGIVHLDLKPENIMCVNK -15.2 ND

PI4KA, PI4KAP2 SGTPMQSAAKAPYLAK -19.4 ND

MER NCMLRDDMTVCVADFGLSKK -19.5 ND

AMPKa1, AMPKa2 DLKPENVLLDAHMNAK -19.9 0

CDK11, CDK8 DLKPANILVMGEGPER -20.4 -0.3

CDK10 DLKVSNLLMTDK -21.1 ND

SRPK1, SRPK2 FVAMKVVK -21.5 ND

PAN3 VMDPTKILITGK -23.6 ND

p70S6K DLKPENIMLNHQGHVK -24.3 3

PHKg2 ATGHEFAVKIMEVTAER -25.2 ND

PDHK1 SPGQPIQVVYVPSHLYHMVFELFKNAMR -25.3 ND

PKCi IYAMKVVK -26.1 1.5

ILK ISMADVKFSFQCPGR -27.6 4.1

ARAF DLKSNNIFLHEGLTVK -39.6 10.8

PIK3CA

RPLWLNWENPDIMSELLFQN

NEIIFKNGDDLRQDMLTLQIIR -41.8 ND

ATR FYIMMCKPK -46.9 1.4

MST4 TQQVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSSYVTK -60 ND

YSK1 EVVAIKIIDLEEAEDEIEDIQQEITVLSQCDSPYITR -67.8 ND


Supplementary Table 2 | Z'-LYTETM Kinase Assay, Invitrogen, Life Technologies.

IC50 (μM) EGFR Her2 TX1-85-1 5.60 2.63 TX2-121-1 >10.0 >10.0

Supplementary Table 3 | Structure activity relationship (SAR) study based on LanthaScreen® technology (Life Technologies) kinase binding assay

Name Structure IC50(nM) Note

Kin001-111 (A-770041) N

N NN

NH

NH2

N

N

O

OO

N

68 Screen hit

Kin001-051 N

N N

O

NH2

85 Screen hit


TX1-85-1 N

N NN

O

NH2

N

N

O

HN

O

23 In main text

TX1-85-1-Biotin N

N NN

NH2

N

N HN

O

HN O

O

O

OO

NH

O

S

HN

NHO

51 In main text

TX1-85-3

N

N

NH2

NN

N

N

O

O

NH

O

207 In main text

TX2-121-1

N

N NN

NH2

N

OHN

NHO

O

49 In main text


TX2-135-2

N

N NN

NH2

N

OHN

NHO

O

326 In main text

TX2-120-1 N

N NN

NH2

N

OHN

O

56 In main text


Supplementary Note:

Materials, Purification, and Analysis

Commercially available reagents and solvents were used without further purification. All reactions were

monitored by thin layer chromatography (TLC) with 0.25 mm E. Merck pre-coated silica gel plates (60

F254) and/or Waters LCMS system (Waters 2489 UV/Visible Detector, Waters 3100 Mass, Waters 515

HPLC pump, Waters 2545 Binary Gradient Module, Waters Reagent Manager, Waters 2767 Sample

Manager) using SunFireTM C18 column (4.6 x 50 mm, 5 µm particle size): solvent A = 0.035% TFA in

Water; solvent B = 0.035% TFA in MeOH; flow rate : 2.5 ml min-1. Purification of reaction products was

carried out by flash chromatography using CombiFlash®Rf with Teledyne Isco RediSep®Rf High

Performance Gold or Silicycle SiliaSepTM High Performance columns (4 g, 12 g or 24 g) and Waters LCMS

system using SunFireTM Prep C18 column (19 x 50 mm, 5 µm particle size): solvent A = 0.035% TFA in

Water; solvent B = 0.035% TFA in MeOH; flow rate: 25 ml min-1. The purity of all compounds was over 95%

and was analyzed with Waters LCMS system. 1H NMR spectra, 13C NMR and 31P NMR were obtained

using a Varian Inova-400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) and/or Inova-600 (600 MHz

for 1H NMR and 150 MHz for 13C) spectrometer. Chemical shifts are reported relative to chloroform (δ =

7.26) for 1H NMR and chloroform (δ = 77.23) for 13C NMR or dimethyl sulfoxide (δ = 2.50) for 1H and

dimethyl sulfoxide (δ = 39.51) for 13C NMR. Data are reported as (br = broad, s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet).


Synthetic Experimental Procedures and Characterization Data

Supplementary Scheme 1

3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10)

To a suspension of 1H-pyrazolo[3,4-d]pyrimidin-4-amine (2.8 g, 20.7 mmol) in DMF (12 mL) was added N-iodosuccinimide (5.59 g, 24.8 mmol) at ambient temperature. The reaction mixture was heated to 80 oC and stirred for 14 hr. The resulting solid was collected by filtration, rinsed with ice-cold ethanol (20 mL), and concentrated in vacuo to provide 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3.9 g, 72%) as an off-white solid. 1H NMR: (600 MHz, CDCl3) δ(ppm): 8.51 (s, 1H); 13C NMR (400 MHz, CDCl3) δ: 157.8; 155.3; 152.2; 103.5; 86.4; MS m/z: 261.92 (M+1).

4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexan-1-one (11)

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10) (531.8 mg, 2.04 mmol) in THF (12 mL) under argon atmosphere were added 1,4-dioxaspiro[4.5]decan-8-ol (826.4 µL, 6.11 mmol), triphenylphosphine (1.07 g, 4.07 mmol) and diisopropyl azodicarboxylate (802.3 µL, 4.07 mmol) at ambient temperature in this order. The reaction mixture was stirred at ambient temperature for 16 hr

NN

I

N

N

NH2

O

NN

I

N

N

NH2

N

NBoc

NH

N

I

N

N

NH2

NH

NN

N

NH2NIS

DMF, 80 oC

72%

O O

OH

PPh3, DIAD

THF, rt;6N

HCl

acetone, rt46%

Na(OAc)3BHDCE, 0

oC → rt

36%

NBoc

HN

10 11 12

NH

N

I

N

N

NH2

NN

I

N

N

NH2

O


and concentrated in vacuo. The crude mixture was taken up in acetone (15 mL) and 6 N HCl (5 mL) was added dropwise. The solution was stirred at ambient temperature for 4 hr and concentrated in vacuo. The residue was purified by flash column chromatography (5% methanol–dichloromethane) to provide 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanone (337.2 mg, 46.3%) as a yellow oil. 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.16 (s, 1H); 4.75 (m, 1H); 3.54 (m, 2H); 3.44 (m, 2H); 2.65 (m, 2H); 2.43 (m, 2H); 13C NMR (400 MHz, CD3OD) δ: 210.2; 158.0; 155.3; 152.6; 103.5; 86.7; 59.1; 40.0; 28.0; MS m/z: 357.90 (M+1).

tert-butyl 4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (12)

To a solution of 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanone (11) (168.5 mg, 0.47 mmol) in 1,2-dichloroethane (2.5 mL) was added tert-butyl piperazine-1-carboxylate (158.2 mg, 0.85 mmol) at 0 oC. After 5 min, sodium triacetoxyborohydride (180.0 mg, 0.849 mmol) was added to the reaction mixture which was warmed up to ambient temperature and stirred for 14 hr. The mixture was diluted with dichloromethane (10 mL), washed with a saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL) and concentrated in vacuo. The residue was purified by flash-column chromatography (10% methanol–dichloromethane) to provide tert-butyl-4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (89.5 mg, 36.0 %) as a white solid. 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.12 (s, 1H); 4.79 (s, 1H); 3.25 (m, 4H); 2.48 (m, 5H); 1.82 (m, 8H); 1.39 (s, 9H); 13C NMR (400 MHz, CDCl3) δ: 161.9; 158.8; 156.6; 152.7; 103.5; 83.9; 73.5; 69.0; 66.6; 60.1; 48.4; 35.2; 31.2; 29.8; 26.0; MS m/z: 528.06 (M+1).

1-(4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethan-1-one (13)

NN

I

N

N

NH2

N

NBoc


NN

I

N

N

NH2

N

NO

To a solution of 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexanone (11) (168.5 mg, 0.47 mmol) in 1,2-dichloroethane (2.5 mL) was added 1-(piperazin-1-yl)ethanone (108.8 mg, 0.85 mmol) at 0 oC. After 5 min, sodium triacetoxyborohydride (180.0 mg, 0.849 mmol) was added to the reaction mixture which was warmed up to ambient temperature and stirred for 14 hr. The mixture was diluted with dichloromethane (10 mL), washed with a saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL) and concentrated in vacuo. The residue was purified by flash-column chromatography (10% methanol–dichloromethane) to 1-(4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethanone (133 mg, 60%). 1H NMR: (400 MHz, CD3OD) δ(ppm): 8.09 (s, 1H); 4.69 (m, 1H); 3.45 (m, 4H); 2.81 (s, 3H); 2.46 (m, 5H); 1.94 (m, 8H); 13C NMR: (400 MHz, CD3OD) δ: 170.5; 159.1; 156.8; 154.3; 104.8; 86.9; 59.9; 56.4; 51.1; 47.5; 42.7; 27.2; 21.3 MS m/z: 470.36 (M+1).


N

N NN

NH2 I

N

NBoc

B(OH)2

NO2

F

N

N NN

NH2

N

NBoc

F

NO2

K2CO3, DMF, 80oC

75%

OH

N

N NN

NH2

N

NBoc

O

NO2

PdCl2(PPh3)2aq. Na2CO3

, dioxane80

oC85%

12 14 15

tert-butyl 4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl) piperazine-1-carboxylate (14)


N

N NN

NH2

N

NBoc

F

NO2

To a solution of tert-butyl 4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (12) (360 mg, 0.68 mmol) in dioxane (6 mL) were added (4-fluoro-3-nitrophenyl)boronic acid (177 mg, 0.96 mmol) and 1 M aqueous solution of sodium carbonate (2 mL) at ambient temperature. The resulting suspension was degassed with argon for 3 min and bis(triphenylphosphine)palladium(II) dichloride (35 mg, 0.05 mmol) was added. The mixture was heated to 80 oC and stirred for 2 hr. It was diluted with dichloromethane (30 mL), washed with water (30 mL) and concentrated in vacuo. The residue was purified by flash-column chromatography (10% methanol–dichloromethane) to provide tert-butyl-4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (312 mg, 85 %). MS m/z: 541.75 (M+1).

tert-butyl 4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl) piperazine-1-carboxylate (15)

N

N NN

NH2

N

NBoc

O

NO2

To a solution of tert-butyl 4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (14) (312 mg, 0.58 mmol) in dimethylformamide (3 ml) were added phenol (81.7 mg 0.87 mmol) and potassium carbonate (120 mg, 0.87 mmol) at ambient temperature. The mixture was heated up to 80 oC and stirred overnight. The mixture was worked up with dichloromethane and water, concentrated in vacuo. The residue was purified by flash column chromatography (10% methanol–dichloromethane) to tert-butyl 4-(4-(4-amino-3-(3-nitro-4-


phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (268.2 mg 75.4%). MS m/z: 615.49 (M+1).

1-(4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethan-1-one (16)

N

N NN

NH2

N

N

F

NO2

O

To a solution of 1-(4-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethan-1-one (13) (234 mg, 0.50 mmol) in dioxane (6 mL) were added (4-fluoro-3-nitrophenyl)boronic acid (136 mg, 0.74 mmol) and 1 M aqueous solution of sodium carbonate (2 mL) at ambient temperature. The resulting suspension was degassed with argon for 3 min and bis(triphenylphosphine)palladium(II) dichloride (47 mg, 0.07 mmol) was added. The mixture was heated to 80 oC and stirred for 2 hr. It was diluted with dichloromethane (30 mL), washed with water (30 mL) and concentrated in vacuo. The residue was purified by flash-column chromatography (10% methanol–dichloromethane) to 1-(4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethanone (188 mg, 78 %). MS m/z: 482.63 (M+1).

1-(4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethan-1-one (17)

N

N NN

NH2

N

N

O

NO2

O


To a solution of 1-(4-(4-(4-amino-3-(4-fluoro-3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethan-1-one (16) (188 mg, 0.39 mmol) in dimethylformamide (3 ml) were added phenol (56.4 mg 0.60 mmol) and potassium carbonate (82.8 mg, 0.60 mmol) at ambient temperature. The mixture was heated up to 80 oC and stirred overnight. The mixture was worked up with dichloromethane and water, concentrated in vacuo. The residue was purified by flash column chromatography (10% methanol–dichloromethane) to 1-(4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethanone (148.7 mg 68.4%). 1H NMR: (400 MHz, CD3OD) δ(ppm): 8.17 (d, J=2 Hz, 1H); 8.11 (s, 1H); 7.78 (dd, J=2 Hz and J=8 Hz, 1H); 7.30 (t, J=8 Hz, 2H); 7.10 (t, J=8 Hz, 1H); 7.05 (d, J=8 Hz, 1H); 6.99 (d, J=8 Hz, 2H); 4.78 (m, 1H); 3.44 (m, 4H); 2.44 (m, 5H); 1.95 (s, 3H); 1.70 (m, 4H); 1.58 (m, 4H); 13C NMR: (400 MHz, CD3OD) δ: 170.0; 158.4; 155.7; 155.1; 153.5; 150.5; 141.3; 133.8; 129.9; 128.6; 125.3; 124.5; 120.8; 118.9; 97.6; 59.6; 53.8; 49.2; 46.1; 41.4; 27.5; 25.3; 19.7; MS m/z: 557.59 (M+1).


Raney Ni,

H2

,

MeOH N

N NN

(Boc)2N

N

NBoc

O

NH2Cl

O

DCM, DMAP, 0oC N

N NN

NH2

N

NH

OHN

O1.

2. TFA : DCM

= 1:1

85%

N

N NN

NH2

N

NBoc

O

NO2

N

N NN

(Boc)2N

N

NBoc

O

NO2

(Boc)2O, DMAP, THF

91% 95%

15 18 19 20

tert-butyl 4-(4-(4-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (18)

N

N NN

(Boc)2N

N

NBoc

O

NO2


To a solution of tert-butyl 4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (15) (268 mg, 0.44 mmol) in tetrahydrofuran (5 mL) were added di-tert-butyl dicarbonate (477 mg, 2.1 mmol) and 4-dimethylaminopyridine (5.3 mg, 0.04 mmol) at ambient temperature. The mixture was stirred for approximately 4 hours, concentrated in vacuo. The residue was purified by flash column chromatography (10% methanol–dichloromethane) to tert-butyl 4-(4-(4-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (326 mg, 91%) MS m/z: 816.07 (M+1).

tert-butyl 4-(4-(3-(3-amino-4-phenoxyphenyl)-4-((tert-butoxycarbonyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (19)

N

N NN

(Boc)2N

N

NBoc

O

NH2

To a solution of tert-butyl 4-(4-(4-((tert-butoxycarbonyl)amino)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (18) (50 mg, 0.06 mmol) in methanol (4 mL) was added Raney nickel (100 mg) which was dispersed in methanol at ambient temperature. The reaction mixture was degassed under vacuum and flushed with hydrogen. This process was repeated three times. The mixture was vigorously stirred under hydrogen balloon for 2 hours, filtered through a celite bed, and washed with methanol quickly three times. All eluted solvent was collected and concentration in vacuo. tert-butyl 4-(4-(3-(3-amino-4-phenoxyphenyl)-4-((tert-butoxycarbonyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate was obtained. (45 mg, 95%) MS m/z: 786.28 (M+1).

N-(5-(4-amino-1-(4-(piperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide (20)


N

N NN

NH2

N

NH

OHN

O

To a solution of tert-butyl 4-(4-(3-(3-amino-4-phenoxyphenyl)-4-((tert-butoxycarbonyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazine-1-carboxylate (19) (60 mg, 0.076 mmol) in dichloromethane (0.5 mL) were added acryloyl chloride (8 µL, 0.10 mmol) and 4-dimethylaminopyridine (4.0 mg, 0.033 mmol) at ambient temperature. The mixture was stirred for 2 hours, treated with trifluoroacetic acid (0.5 mL), stirred for additional 2 hours at ambient temperature, and concentrated. N-(5-(4-amino-1-(4-(piperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide was obtained (35 mg, 85%) MS m/z: 540.07 (M+1).

tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (21)

To a solution of 1-(4-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)cyclohexyl)piperazin-1-yl)ethanone (17) (148.7 mg, 0.267 mmol) in tetrahydrofuran (5 mL) were added di-tert-butyl dicarbonate (238 mg, 1.1 mmol) and 4-dimethylaminopyridine (5.3 mg, 0.04 mmol) at ambient temperature. The mixture was stirred for approximately 4 hours, concentrated in vacuo. The residue was purified by flash column chromatography (10% methanol–dichloromethane) to tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (188 mg, 93%) MS m/z: 757.91 (M+1).

N

N NN

(Boc)2N

N

N

O

NO2

O


tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (22)

N

N NN

(Boc)2N

N

N

O

NH2

O

To a solution of tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (21) (50 mg, 0.066 mmol) in methanol (4 mL) was added Raney nickel (100 mg) which was dispersed in methanol at ambient temperature. The reaction mixture was degassed under vacuum and flushed with hydrogen. This process was repeated three times. The mixture was vigorously stirred under hydrogen baloon for 2 hours, filtered through a celite bed, and washed with methanol quickly three times. All elute solvent was collected and concentration in vacuo. tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate was obtained (48 mg, 98%) MS m/z: 728.04 (M+1).

N-(5-(4-amino-1-(4-(piperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide (TX1-85-1) (4)

N

N NN

NH2

N

N

OHN

O

O

To a solution of tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (22) (30 mg, 0.041 mmol) in dichloromethane (0.5 mL) were


added acryloyl chloride (4 µL, 0.05 mmol) and 4-dimethylaminopyridine (4.0 mg, 0.033 mmol) at ambient temperature. The mixture was stirred for 2 hours, treated with trifluoroacetic acid (0.5 mL), stirred for additional 2 hours at ambient temperature, and concentrated. The crude mixture was diluted with dimethyl sulfoxide (1 mL) and directly purified by preparative reverse-phase HPLC (methanol/water gradient) to provide N-(5-(4-amino-1-(4-(piperazin-1-yl)cyclohexyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide as a TFA salt. (TX1-85-1) (12 mg, 50%) 1H NMR: (400 MHz, DMSO-d6) δ(ppm): 9.98 (s, 1H); 8.32 (d, J=2 Hz, 1H); 8.20 (s, 1H); 7.40 (m, 3H); 7.16 (t, J=7.2 Hz, 1H); 7.09 (d, J=7.2 Hz, 2H); 7.01 (d, J=7.2 Hz, 1H); 6.66 (dd, J=12.8 and 10 Hz, 1H); 6.24 (dd, J=12.8 and 2 Hz, 1H); 5.73 (dd, J=10 and 2 Hz, 1H); 4.62 (m, 1H); 3.41 (br m, 4H); 2.48 (br m, 5H); 2.44 (s, 3H); 1.96 (m, 4H); 1.45 (br m, 4H); 13C NMR: (400 MHz, DMSO-d6) δ: 170.0; 164.4; 158.5; 156.8; 155.9; 154.0; 148.3; 143.0; 132.0; 130.4; 129.9; 128.6; 124.3; 124.0; 119.6; 119.3; 97.8; 62.1; 55.6; 48.9; 46.6; 41.7; 31.3; 27.1; 21.6; MS m/z: 581.43 (M+1).

N-(5-(1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)propionamide (TX1-85-3) (9)

N

N NN

NH2

N

N

OHN

O

O

To a solution of tert-butyl (1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-3-(3-amino-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (22) (30 mg, 0.041 mmol) in dichloromethane (0.5 mL) were added propionyl chloride (4 µL, 0.05 mmol) and 4-dimethylaminopyridine (4.0 mg, 0.033 mmol) at ambient temperature. The mixture was stirred for 2 hours, treated with trifluoroacetic acid (0.5 mL), stirred for additional 2 hours at ambient temperature, and concentrated. The crude mixture was diluted with dimethyl sulfoxide (1 mL) and directly purified by preparative reverse-phase HPLC (methanol/water gradient) to provide N-(5-(1-(4-(4-acetylpiperazin-1-yl)cyclohexyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)propionamide as a TFA salt (TX1-85-3) (9mg, 38%) 1H-NMR: (400 MHz, DMSO-d6) δ(ppm): 9.68 (s, 1H); 8.20 (s, 1H); 8.19 (s, 1H); 7.38 (m, 3H); 7.15 (t, J=7.2 Hz, 1H); 7.05 (m, 3H); 4.81 (m, 1H); 2.50 (m, 5H); 2.33 (s, 3H); 1.96 (m, 4H); 1.73 (br m, 4H); 13C NMR: (400 MHz, DMSO-d6) δ: 173.3; 168.4; 158.5; 156.9; 154.1; 148.0; 142.8; 130.3; 128.8; 124.3; 124.0; 120.0; 118.9; 97.7; 62.1; 59.0; 54.2; 50.3; 46.8; 40.8; 29.5; 27.6; 26.2; 21.5; 10.1; MS m/z: 583.28 (M+1).



B(OH)2

NO2

F

N

N NN

NH2

N

F

NO2

N

N NN

NH2

N

O

NO2OH

K2CO3PdCl2(PPh3)2aq. Na2CO3

, dioxane80

oC

N

N NH

N

NH2 I NBoc

Br

K2CO3

N

N NN

NH2 I

NBoc

HCl N

N NN

NH2 I

NH

Br NHBoc

N

N NN

NH2 I

N

NHBoc

K2CO3 DMF

67%

NHBoc NHBoc

54%

58% over 2

steps

23

10

24 25

26 27

tert-butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (23)

N

N NN

NH2 I

NBoc

To a solution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10) (1g, 3.83 mmol) in dimethylformamide (15 ml) were added tert-butyl 4-bromopiperidine-1-carboxylate (1.5 g, 5.68 mmol) and potassium carbonate (1g, 7.25 mmol) at ambient temperature. The mixture was heated up to 80 oC and stirred overnight. The mixture was worked up with dichloromethane and water, concentrated in vacuo. The residue was purified by flash column chromatography (8% methanol–dichloromethane) to tert-butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (918.5 mg 53.8%). 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.20 (s, 1H); 4.21 (m, 1H); 3.03 (m, 4H); 2.10 (m, 2H); 1.95 (m, 2H); 1.49 (s, 9H); MS m/z: 445.76 (M+1).

3-iodo-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (24)


N

N NN

NH2 I

NH

To a solution of tert-butyl 4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (23) in dichloromethane (10ml) were added HCl (1 M, 2ml) at ambient temperature for 4 hours. The mixture were neutralized by sodium bicarbonate and worked up with dichloromethane, concentrated in vacuo to afford 3-iodo-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (697 mg, 98%) without further purification. 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.11 (s, 1H); 5.39 (s, 1H); 4.13 (m, 1H); 3.56 (m, 2H); 2.90 (m, 2H); 1.98 (m, 2H); 1.83 (m, 2H); 13C NMR (400 MHz, CD3OD) δ: 157.7; 155.0; 152.6; 103.5; 87.0; 66.7; 30.9; 27.2; MS m/z: 345.54 (M+1).

tert-butyl (2-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate (25)

N

N NN

NH2 I

N

NHBoc

To a solution of 3-iodo-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (24) (100 mg, 0.30 mmol)

in dimethylformamide (3 mL) were added tert-butyl (2-bromoethyl)carbamate (78 mg, 0.35 mmol) and

potassium carbonate (128 mg, 0.93 mmol) at ambient temperature. The reaction mixture was heated to

80 oC, stirred at ambient temperature overnight. The crude was worked up with dichloromethane and

water. The product was purified by flash column chromatography (8% methanol–dichloromethane) to

provide tert-butyl (2-(4-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-

yl)ethyl)carbamate (98.4 mg, 67.2%). 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.11 (s, 1H); 8.02 (s, 1H); 4.59

(m, 1H); 3.16 (m, 2H); 3.03 (m, 2H); 2.47 (m, 2H); 2.20 (m, 4H); 1.87 (m, 2H); 1.38 (s, 9H); 13C NMR (400

MHz, CD3OD) δ: 161.7; 157.9; 155.3; 152.7; 103.6; 86.5; 78.7; 57.1; 54.8; 52.3; 37.3; 30.7; 27.4; MS m/z:

488.64(M+1).

tert-butyl (2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate (27)


N

N NN

NH2

N

O

NO2

NHBoc

Repeating the reaction procedures mentioned above, tert-butyl (2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate (67.0mg, 58.4%) was obtained as a yellow solid. 1H NMR: (600 MHz, CD3OD) δ(ppm): 8.68 (s, 1H); 8.23 (s, 1H); 8.19 (s, 1H); 7.83 (dd, J=9 Hz and 2.4 Hz, 1H); 7.37 (m, 2H); 7.17 (t, J=9 Hz, 1H); 7.10 (d, J=9 Hz, 1H); 7.05 (m, 2H); 7.01 (m, 1H); 4.68 (m, 1H); 3.92 (m, 2H); 3.20 (m, 2H); 3.05 (m, 2H); 2.49 (m, 2H); 2.30 (m, 2H); 2.22 (m, 2H); 1.93 (m, 2H); 1.41 (s, 9H); 13C NMR (400 MHz, CD3OD) δ: 162.7; 158.3; 155.5; 155.2; 153.6; 150.5; 141.7; 141.3; 133.7; 129.9; 128.4; 125.2; 124.5; 120.7; 118.9; 105.0; 97.9; 78.6; 63.3; 57.2; 54.3; 52.4; 37.3; 30.7; 27.5; 23.9; MS m/z: 575.62 (M+1).



Cl

O

N

N NN

NH2

N

OHN

NH

O

O

N

N NN

NH2

N

O

NO2

NHO

H2, Raney

Ni

N

N NN

(Boc)2N

N

O

NH2

NHO

(Boc)2O, DMAPN

N NN

(Boc)2N

N

O

NO2

NHO

1.

2. TFA, DCM

N

N NN

NH2

N

O

NO2

NHBoc

O

OH

EDCI, DMAP, DIEA, DMF

50%

1. TFA, DCM

2.

27

28 29

30 TX2-121-1 (6)

(R)-4-((3R,5R,7R)-adamantan-1-yl)-N-(2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-2-methylbutanamide (28)


N

N NN

NH2

N

O

NO2

NHO

To a solution of tert-butyl (2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)carbamate (27) (30 mg, 0.05 mmol) in dichloromethane (1.2 mL) was added trifluoroacetic acid (500 µL) at ambient temperature. The reaction mixture was stirred at ambient temperature for 2 hr and concentrated in vacuo. The crude product was taken up in dimethylformamide (1.0 mL), treated with (2R)-4-((1r,3S)-adamantan-1-yl)-2-methylbutanoic acid (10 mg, 0.042 mmol), N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (30.8 mg, 0.16 mmol) and 4-(dimethylamino)pyridine (4 mg, 0.03 mmol) at ambient temperature in this order. The reaction mixture was stirred at ambient temperature for 3 hr. The residue was purified by flash column chromatography (methanol–dichloromethane) to afford (R)-4-((3R,5R,7R)-adamantan-1-yl)-N-(2-(4-(4-amino-3-(3-nitro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-2-methylbutanamide (17.4 mg, 50.2%). MS m/z: 693.45 (M+1).

(R)-N-(2-(4-(3-(3-acrylamido-4-phenoxyphenyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-4-((3R,5R,7R)-adamantan-1-yl)-2-methylbutanamide (TX2-121-1) (6)


N

N NN

NH2

N

OHN

NH

O

O

Repeating the reaction procedures mentioned above, (R)-N-(2-(4-(3-(3-acrylamido-4-phenoxyphenyl)-4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-4-((3R,5R,7R)-adamantan-1-yl)-2-methylbutanamide (7.3 mg) (TX2-121-1) was obtained and purified by preparative reverse-phase HPLC (methanol/water gradient). 1H NMR: (600 MHz, DMSO-d6) δ(ppm): 9.94 (s, 1H); 8.33 (s, 1H); 8.22 (s, 1H); 7.37 (t, J=8.4Hz, 2H); 7.33 (dd, J=8.4 Hz and 1.8 Hz, 1H ); 7.11 (d, J=8.4 Hz, 1H); 7.07 (d, J=8.4 Hz, 2H); 6.96 (d, J=8.4Hz, 1H); 6.64 (dd, J=17.4 and 10.8 Hz, 1H); 6.19 (dd, J=17.4 and 1.8 Hz, 1H); 5.69 (dd, J=10.8 and 1.8 Hz, 1H); 4.91 (s, 1H); 3.42 (br m, 5H); 2.56 (br m, 4H); 2.39 (s, 3H); 2.05 (m, 4H); 1.91 (br m, 4H);

13C NMR (400 MHz, CD3OD) δ: 177.0; 164.4; 158.5; 157.7; 155.8; 154.2; 153.9; 144.0; 142.6; 141.5; 130.2; 128.6; 123.0; 120.9; 119.3; 117.5; 116.6; 115.7; 97.8; 57.4; 54.4; 52.8; 42.3; 42.0; 40.9; 37.1; 36.7; 32.0; 31.5; 28.5; 27.3; 22.7; 18.5; MS m/z: 717.42 (M+1).

(R)-4-((3R,5R,7R)-adamantan-1-yl)-N-(2-(4-(4-amino-3-(4-phenoxy-3-propionamidophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-2-methylbutanamide (TX2-135-2) (7)


N

N NN

NH2

N

OHN

NH

O

O

Repeating the reaction procedures mentioned above, (R)-4-((3R,5R,7R)-adamantan-1-yl)-N-(2-(4-(4-amino-3-(4-phenoxy-3-propionamidophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethyl)-2-methylbutanamide (10.2 mg) (TX2-135-2) was obtained and purified by preparative reverse-phase HPLC (methanol/water gradient). 1H NMR: (400 MHz, DMSO-d6) δ(ppm): 9.99 (s, 1H); 8.32 (s, 1H); 8.20 (s, 1H); 7.66 (t, 2H); 7.39 (dd, 1H ); 7.35 (m, 1H); 7.06 (d, 2H); 6.88 (d, 1H); 4.62 (s, 1H); 3.39 (br m, 5H); 2.99 (br m, 4H); 2.38 (s, 3H); 1.84 (m, 4H); 1.57 (br m, 4H); 13C NMR (400 MHz, CD3OD) δ: 176.4; 162.1; 158.7; 157.5; 156.8; 153.6; 143.3; 142.5; 141.3; 128.6; 121.9; 104.3; 97.4; 57.3; 55.3; 52.6; 43.3; 42.5; 41.7; 37.5; 36.6; 32.4; 31.8; 29.5; 28.4; 22.4; 20.4; 18.3; 10.2; MS m/z: 719.21 (M+1).

N-(5-(4-amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide (5.0 mg) (TX2-120-1) (8)

N

N NN

NH2

N

OHN

O

Repeating the reaction procedures mentioned above, N-(5-(4-amino-1-(1-methylpiperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-2-phenoxyphenyl)acrylamide (5.0 mg) (TX2-120-1) was obtained and purified by preparative reverse-phase HPLC (methanol/water gradient). 1H NMR: (600 MHz, DMSO-d6) δ(ppm): 10.0 (s, 1H); 9.56 (br s, 1H); 8.42 (s, 1H); 8.30 (s, 1H); 7.473 (t, J=8.4 Hz, 2H); 7.39 (dd, J=8.4 Hz and 1.8 Hz, 1H); 7.19 (t, J=8.4 Hz, 1H); 7.11 (d, J=8.4 Hz, 2H); 7.03 (d, J=8.4 Hz, 1H); 6.70 (dd, J=17.4 and


10.8 Hz, 1H); 6.26 (dd, J=17.4 and 1.8 Hz, 1H); 5.76 (dd, J=10.8 and 1.8 Hz, 1H); 5.05 (br m, 1H); 3.80 (m); 3.70 (br m); 3.16 (br m); 2.49 (s); MS m/z: 470.34 (M+1).


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