Supplementary InformationTitle: Structure Activity Relationships of Human Galactokinase Inhibitors

Authors: Li Liua, Manshu Tangb, Martin J. Walsha,c, Kyle R. Brimacombea, Rajan Pragania,

Cordelle Tanegaa, Jason M. Rohdea, Heather L. Bakera, Elizabeth Fernandeza, Burchelle

Blackmana,e, James M. Bougiea, William Leistera, Douglas S. Aulda,d, Min Shena, Kent Laib,

Matthew B. Boxera*

aNational Center for Advancing Translational Sciences, National Institutes of Health, 9800

Medical Center Drive, Rockville, MD, 20850, USA

b Division of Medical Genetics, Department of Pediatrics, University of Utah, Rm 2C412 SOM,

50 N. Medical Drive, Salt Lake City, UT 84132.cPresent address: Dow AgroSciences LLC, Crop-Protection Discovery, Group, Bldg. 306/E2/980, 9330 Zionsville Road, Indianapolis, IN, 46268, USA.

dPresent address: Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research,

Cambridge, MA, 02139, USA.

ePresent address: Imaging Probe Development Center, National Heart, Lung, and Blood Institute,

National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850 USA

* To whom correspondence should be addressed:

Matthew Boxer, Ph.D.9800 Medical Center Dr.Rockville, MD 20850Phone: 301-217-4681. Fax: 301-217-5736. Email: boxerm@mail.nih.gov

S1

Table of Contents

Biology/ScreeningHTS Protocol and Analysis; Page S2Kinase-Glo Plus Counterscreen protocol; Page S2Phenol-HRP redox Counterscreen protocol; Page S3GALK substrate competition protocol; Page S3-4Cell based Galactose-1-phosphate accumulation assay; Page S4Kinome Scan results; Pages S9 – 19

ChemistryGeneral methods (chemistry); Pages S4Synthesis and characterization of analog 19; Pages S5-S7HRMS Table for all analogs; Page S8X-ray Structure experimental details and results for compound 32; Page S20-S31

HTS Protocol and analysis. The high-throughput inhibitor screen was performed by coupling the activity of recombinant human GALK1 to the Kinase-Glo Plus luminescent ATP detection kit, using ATP depletion as a measure of GALK1 turnover. Specifically, three μL/well of ATP substrate solution (35 μM ATP) in assay buffer (20 mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was dispensed into 1536-well assay plates (Greiner, white solid-bottom medium-binding plates). 23 nL of compound (solubilized in DMSO) were transferred to the assay plates using a Kalypsys 1536-well pintool. One μL/well of GALK-galactose solution (5 nM GALK1, 100 μM galactose) in assay buffer was then added, yielding a final reaction volume of 4 μL/well. Following a 1 hour room temperature incubation, 4 μL of Kinase-Glo Plus detection reagent was added to provide an ATP-dependent luminescent readout (final assay volume: 8 μL/well). Luminescence was detected using a ViewLux plate reader (PerkinElmer, Waltham, MA) after a10 minute incubation, using a 1 second exposure time and 2x binning.

All concentration response curves (CRCs) were fitted using in-house developed software (http://ncgc.nih.gov/pub/openhts/). Curves were categorized into four classes: complete response curves (Class 1), partial curves (Class 2), single point actives (Class 3) and inactives (Class 4). Compounds with the highest quality, Class 1 and Class 2 curves, were prioritized for follow-up.

Kinase-Glo Plus Counterscreen protocol. To identify compounds with potential inhibitory activity against the detection reagent used in the screen, a counterscreen was run to monitor the conversion of ATP to a luminescent Kinase-Glo Plus readout in the absence of GALK. Four μL of ATP substrate solution (10 μM ATP) in assay buffer (50 mM imidazole pH 7.2, 50 mM KCl, 7 mM MgCl2, 0.01% Tween 20, 0.05% BSA, final concentration) was dispensed into white 1536-well plates. 23 nL of compound was delivered by pintool, and 2 μL of Kinase-Glo Plus detection reagent was added. Following an 8 minute incubation at room temperature, luminescence was measured by ViewLux using a 5 second exposure time. Data were normalized

S2

to the controls for basal activity (DMSO only) and 100% inhibition (40 μM SIB 1757) and curve fitting was performed using Hill equation.Phenol-HRP redox Counterscreen protocol. One mM dithiothreitol (DTT) was used as a reducing agent to maintain GALK in its active form in the qHTS screen. However in the presence of DTT, it has been shown that a number of compounds can undergo redox cycling, generating a significant amount of H2O2 that can oxidize crucial cysteine residues in enzymes, leading to false-positive results. To address this, a redox counterscreen was run to identify compounds capable of producing H2O2 in a buffered assay system, using horseradish peroxidase (HRP) –mediated oxidation of phenol red as a readout.

Two μL/well of GALK buffer (20 mM HEPES pH 8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA, final concentration) was dispensed into 1536-well assay plates (Greiner, black clear bottom plates). Compound and control solutions (23 nL each) were transferred by pintool, and plates were incubated at room temperature for 30 minutes. Presence of H2O2 produced by redox cycling compounds was detected by adding 2 μL/well phenol red - HRP detection reagent (100 μL/mL phenol red in 1x Hanks Balanced Salt Solution, 25 U/mL HRP, final concentration). After 15 minutes of room temperature incubation, 2 μL/well 1N NaOH was added to stop the reaction. Plates were then incubated for an additional 1 hour at room temperature. The absorbance at 600 nm was measured using a ViewLux plate reader (6000 light energy, 2 second exposure, and 2x binning).

GALK substrate competition protocol. The mechanism of GALK inhibition was tested for a subset of compounds, using substrate competition to examine whether compounds competed with galactose or ATP for binding. Competition was assayed by coupling production of ADP by GALK to rabbit pyruvate kinase (PyK), which converts phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP. This reaction is additionally coupled to lactate dehydrogenase (LDH), which converts pyruvate and NADH to lactate and NAD+, respectively. Conversion of NADH was monitored kinetically by measuring NADH fluorescence at ex340/em450. Briefly, a substrate master mix consisting of 50 nM GALK, saturating concentrations of a single substrate (2 mM ATP or 1 mM galactose), excess rabbit PyK, PEP and LDH (500 nM, 0.5 mM and 500 nM, respectively), and 0.18 mM NADH was prepared. Serial dilutions of the competing substrate (galactose or ATP) and small molecule inhibitor were also prepared, spanning their respective Kms and IC50s. All reagents were prepared in HEPES buffer, pH 8.0, containing 5 mM MgCl2, 60 mM NaCl, 1 mM DTT and 0.01% BSA. Individual aliquots of substrate mix, competing substrate and inhibitor were then mixed to begin the reaction, which was immediately transferred to a black multiwell plate and measured on a ViewLux reader, with kinetic change in NADH fluorescence being measured every 15 seconds. Changes in fluorescence were plotted for each pair of substrate and inhibitor concentrations to determine initial velocities, which were then transformed to Lineweaver-Burk plots using Prism graphical software (GraphPad Software, Inc.; La Jolla, CA) for the demonstration of binding site competition.

Gal-1-p Accumulation Assay. Skin fibroblasts derived from GALT-deficient patients were maintained in galactose-free culture medium supplemented with 10% hexose-free fetal bovine serum (FBS). Before galactose challenge, inhibitors were added to the medium at designated concentrations and incubated at 37 ºC for 4 hr. Then, galactose was added to reach 0.05% in the medium. After 4 hr of challenge, cells were collected and washed twice with PBS. Then, the

S3

cells were disrupted in 300 µL of ice cold hypotonic buffer containing 25 mM Tris–HCl (pH 7.4), 25 mM NaCl, 0.5 mM EDTA, and protease inhibitor cocktail (Roche, cat. # 11 697 498 001). The lysates were passed five times through a 30 gauge needle and centrifuged for 20 min at 16,000 x g and 4 ºC. A small portion of supernatant was saved for protein concentration measurement. Gal-1-p level was measured using the alkaline phosphatase coupled method previously described. The gal-1-p concentration was normalized to protein concentration7.

General Methods for Chemistry. All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylforamide (DMF), acetonitrile, dioxane, dimethoxyethane, methanol and triethylamine were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system using a Phenomenex Luna C18 column (5 micron, 30 x 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was S3 used during the purification. Fraction collection was triggered by UV detection (220 nm). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Final QC LCMS Method: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 x 75 mm) was used at a temperature of 50° C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1, Method 2 and Method 3. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with undeuterated solvent (DMSO-d6 at 2.49 ppm) as internal standard for DMSO-d6 solutions. All of the analogs tested in the biological assays have purity greater than 95%, based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02)

S4

Synthesis and Characterization of 19

19. 2-(benzo[d]oxazol-2-ylamino)-4-(2-chlorophenyl)-3,4,7,8-tetrahydroquinazolin-5(6H)-oneThe mixture of cyclohexane-1,3-dione (0.1 g, 0.892 mmol), 2-chlorobenzaldehyde (0.100 ml, 0.892 mmol) and 1-(benzo[d]oxazol-2-yl)guanidine (0.105 g, 0.595 mmol) was heated at 120 °C for 1 hr in a sealed tube. DMF (~2 mL) was added to the hot mixture to dissolve the residue. The crude product was purified by reverse phase chromotography (5-100% gradient of acetonitrile/H2O) to give 0.13g (55.7%) of product as white solid.

1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.37 (d, J = 2.7 Hz, 1H), 7.53 – 7.48 (m, 1H), 7.44 – 7.40 (m, 1H), 7.40 – 7.36 (m, 1H), 7.32 (tdd, J = 5.8, 3.3, 1.3 Hz, 3H), 7.19 (tt, J = 7.8, 1.3 Hz, 1H), 7.12 (tt, J = 7.6, 1.3 Hz, 1H), 5.89 (d, J = 2.7 Hz, 1H), 2.76 – 2.58 (m, 2H), 2.33 (q, J = 5.7, 4.6 Hz, 2H), 2.10 – 1.99 (m, 2H); 13C NMR (400 MHz, DMSO-d6) δ193.61, 164.92, 154.55, 151.26, 147.17, 141.60, 139.17, 132.67, 130.40, 130.23, 128.93, 128.24, 124.18, 122.83, 116.89, 109.72, 106.49, 49.43, 36.69, 26.26, 21.08; HRMS: m/z (M+H)+ =393.1118 (Calculated for C21H18ClN4O2 = 393.1124).

S5

1H NMR of 19

S6

13C NMR of 19

S7

HRMS data

NCGC#final QC

retention time found cal Molecular formula1 NCGC00187645-01 6.895 379.2144 379.2145 C22H27N4O22 NCGC00188035-01 6.869 413.1988 413.1989 C25H25N4O23 NCGC00187642-03 5.847 337.1664 337.1660 C19H21N4O24 NCGC00188036-01 5.522 323.1515 323.1514 C18H19N4O25 NCGC00186052-01 6.375 351.1822 351.1827 C20H23N4O26 NCGC00187643-01 5.074 311.1513 311.1514 C17H19N4O27 NCGC00187641-01 5.468 323.1509 323.1514 C18H19N4O28 NCGC00188583-01 4.024 350.1984 350.1987 C20H24N5O9 NCGC00188582-01 4.684 426.2285 426.2288 C26H28N5O

10 NCGC00188843-01 5.221 359.1512 359.1514 C21H19N4O211 NCGC00188849-01 5.341 377.1415 377.1420 C21H18FN4O212 NCGC00185792-02 5.676 393.1118 393.1117 C21H18ClN4O213 NCGC00188836-01 5.186 389.162 389.1620 C22H21N4O314 NCGC00188842-01 5.500 373.1668 373.1670 C22H21N4O215 NCGC00188850-01 5.476 373.1658 373.1657 C22H21N4O216 NCGC00188835-01 5.312 377.1411 377.1406 C21H18FN4O217 NCGC00188844-01 5.643 393.1112 393.1113 C21H18ClN4O218 NCGC00188847-01 5.248 389.1615 389.1620 C22H21N4O319 NCGC00188571-02 5.677 393.1118 393.1124 C21H18ClN4O220 NCGC00188851-01 5.489 389.1612 389.1620 C22H21N4O321 NCGC00189071-01 5.793 437.0628 437.0619 C21H18BrN4O222 NCGc00188845-01 6.000 427.1389 427.1377 C22H18F3N4O223 NCGC00189068-01 5.738 427.0736 427.0735 C21H17Cl2N4O224 NCGC00188839-01 3.555 180.5767 180.5770 C20H18N5O225 NCGC00188840-01 3.548 360.1462 360.1455 C20H18N5O226 NCGC00229725-01 4.052 349.141 349.1411 C18H17N6O227 NCGC00188837-01 4.888 394.1068 394.1065 C20H17ClN5O228 NCGC00189072-01 4.969 394.1072 394.1077 C20H17ClN5O229 NCGC00189281-01 5.092 438.0562 438.0558 C20H17BrN5O230 NCGC00189284-01 5.148 438.057 438.0572 C20H17BrN5O231 NCGC00189285-01 4.984 438.057 438.0567 C20H17BrN5O232 NCGC00189069-01 5.434 428.1327 428.133 C21H17F3N5O233 NCGC00189070-01 5.522 428.1338 428.1338 C21H17F3N5O234 NCGC00189286-01 4.577 427.0519 427.0516 C18H16BrN6O235 NCGC00189288-01 4.817 441.0675 441.0672 C19H18BrN6O236 NCGC00238624-07 4.560 405.0836 405.0837 C18H15ClN6O2Na37 NCGC00250068-01 4.838 397.1183 397.1174 C19H18ClN6O238 NCGC00189282-01 4.809 397.1178 397.1186 C19H18ClN6O2

Kinome Scan

S8

Compound Name Ambit Gene SymbolEntrez Gene

Symbol%

Control Compd. Conc. (nM)

S9

NCGC00238624-03 AAK1 AAK1 100 10000NCGC00238624-03 ABL1(E255K)-phosphorylated ABL1 100 10000

NCGC00238624-03ABL1(F317I)-nonphosphorylated ABL1 80 10000

NCGC00238624-03 ABL1(F317I)-phosphorylated ABL1 82 10000

NCGC00238624-03ABL1(F317L)-nonphosphorylated ABL1 90 10000

NCGC00238624-03 ABL1(F317L)-phosphorylated ABL1 98 10000

NCGC00238624-03ABL1(H396P)-nonphosphorylated ABL1 86 10000

NCGC00238624-03 ABL1(H396P)-phosphorylated ABL1 82 10000

NCGC00238624-03ABL1(M351T)-phosphorylated ABL1 68 10000

NCGC00238624-03ABL1(Q252H)-nonphosphorylated ABL1 100 10000

NCGC00238624-03ABL1(Q252H)-phosphorylated ABL1 94 10000

NCGC00238624-03ABL1(T315I)-nonphosphorylated ABL1 65 10000

NCGC00238624-03 ABL1(T315I)-phosphorylated ABL1 80 10000NCGC00238624-03 ABL1(Y253F)-phosphorylated ABL1 87 10000NCGC00238624-03 ABL1-nonphosphorylated ABL1 86 10000NCGC00238624-03 ABL1-phosphorylated ABL1 100 10000NCGC00238624-03 ABL2 ABL2 100 10000NCGC00238624-03 ACVR1 ACVR1 100 10000NCGC00238624-03 ACVR1B ACVR1B 100 10000NCGC00238624-03 ACVR2A ACVR2A 100 10000NCGC00238624-03 ACVR2B ACVR2B 90 10000NCGC00238624-03 ACVRL1 ACVRL1 100 10000NCGC00238624-03 ADCK3 CABC1 100 10000NCGC00238624-03 ADCK4 ADCK4 100 10000NCGC00238624-03 AKT1 AKT1 100 10000NCGC00238624-03 AKT2 AKT2 86 10000NCGC00238624-03 AKT3 AKT3 100 10000NCGC00238624-03 ALK ALK 100 10000NCGC00238624-03 AMPK-alpha1 PRKAA1 100 10000NCGC00238624-03 AMPK-alpha2 PRKAA2 100 10000NCGC00238624-03 ANKK1 ANKK1 69 10000NCGC00238624-03 ARK5 NUAK1 100 10000NCGC00238624-03 ASK1 MAP3K5 100 10000NCGC00238624-03 ASK2 MAP3K6 88 10000NCGC00238624-03 AURKA AURKA 100 10000NCGC00238624-03 AURKB AURKB 100 10000NCGC00238624-03 AURKC AURKC 100 10000NCGC00238624-03 AXL AXL 72 10000

S10

NCGC00238624-03 BIKE BMP2K 83 10000NCGC00238624-03 BLK BLK 100 10000NCGC00238624-03 BMPR1A BMPR1A 100 10000NCGC00238624-03 BMPR1B BMPR1B 80 10000NCGC00238624-03 BMPR2 BMPR2 87 10000NCGC00238624-03 BMX BMX 96 10000NCGC00238624-03 BRAF BRAF 100 10000NCGC00238624-03 BRAF(V600E) BRAF 100 10000NCGC00238624-03 BRK PTK6 100 10000NCGC00238624-03 BRSK1 BRSK1 100 10000NCGC00238624-03 BRSK2 BRSK2 100 10000NCGC00238624-03 BTK BTK 93 10000NCGC00238624-03 CAMK1 CAMK1 100 10000NCGC00238624-03 CAMK1D CAMK1D 100 10000NCGC00238624-03 CAMK1G CAMK1G 100 10000NCGC00238624-03 CAMK2A CAMK2A 100 10000NCGC00238624-03 CAMK2B CAMK2B 95 10000NCGC00238624-03 CAMK2D CAMK2D 100 10000NCGC00238624-03 CAMK2G CAMK2G 100 10000NCGC00238624-03 CAMK4 CAMK4 100 10000NCGC00238624-03 CAMKK1 CAMKK1 100 10000NCGC00238624-03 CAMKK2 CAMKK2 100 10000NCGC00238624-03 CASK CASK 100 10000NCGC00238624-03 CDC2L1 CDC2L1 100 10000NCGC00238624-03 CDC2L2 CDC2L2 100 10000NCGC00238624-03 CDC2L5 CDC2L5 100 10000NCGC00238624-03 CDK11 CDC2L6 100 10000NCGC00238624-03 CDK2 CDK2 100 10000NCGC00238624-03 CDK3 CDK3 100 10000NCGC00238624-03 CDK4-cyclinD1 CDK4 100 10000NCGC00238624-03 CDK4-cyclinD3 CDK4 93 10000NCGC00238624-03 CDK5 CDK5 100 10000NCGC00238624-03 CDK7 CDK7 55 10000NCGC00238624-03 CDK8 CDK8 87 10000NCGC00238624-03 CDK9 CDK9 100 10000NCGC00238624-03 CDKL1 CDKL1 57 10000NCGC00238624-03 CDKL2 CDKL2 79 10000NCGC00238624-03 CDKL3 CDKL3 89 10000NCGC00238624-03 CDKL5 CDKL5 100 10000NCGC00238624-03 CHEK1 CHEK1 100 10000NCGC00238624-03 CHEK2 CHEK2 100 10000NCGC00238624-03 CIT CIT 35 10000NCGC00238624-03 CLK1 CLK1 77 10000NCGC00238624-03 CLK2 CLK2 71 10000

S11

NCGC00238624-03 CLK3 CLK3 90 10000NCGC00238624-03 CLK4 CLK4 94 10000NCGC00238624-03 CSF1R CSF1R 100 10000NCGC00238624-03 CSK CSK 100 10000NCGC00238624-03 CSNK1A1 CSNK1A1 100 10000NCGC00238624-03 CSNK1A1L CSNK1A1L 100 10000NCGC00238624-03 CSNK1D CSNK1D 61 10000NCGC00238624-03 CSNK1E CSNK1E 34 10000NCGC00238624-03 CSNK1G1 CSNK1G1 100 10000NCGC00238624-03 CSNK1G2 CSNK1G2 100 10000NCGC00238624-03 CSNK1G3 CSNK1G3 100 10000NCGC00238624-03 CSNK2A1 CSNK2A1 100 10000NCGC00238624-03 CSNK2A2 CSNK2A2 91 10000NCGC00238624-03 CTK MATK 100 10000NCGC00238624-03 DAPK1 DAPK1 100 10000NCGC00238624-03 DAPK2 DAPK2 100 10000NCGC00238624-03 DAPK3 DAPK3 100 10000NCGC00238624-03 DCAMKL1 DCLK1 100 10000NCGC00238624-03 DCAMKL2 DCLK2 100 10000NCGC00238624-03 DCAMKL3 DCLK3 100 10000NCGC00238624-03 DDR1 DDR1 100 10000NCGC00238624-03 DDR2 DDR2 100 10000NCGC00238624-03 DLK MAP3K12 55 10000NCGC00238624-03 DMPK DMPK 85 10000NCGC00238624-03 DMPK2 CDC42BPG 97 10000NCGC00238624-03 DRAK1 STK17A 87 10000NCGC00238624-03 DRAK2 STK17B 95 10000NCGC00238624-03 DYRK1A DYRK1A 93 10000NCGC00238624-03 DYRK1B DYRK1B 99 10000NCGC00238624-03 DYRK2 DYRK2 89 10000NCGC00238624-03 EGFR EGFR 82 10000NCGC00238624-03 EGFR(E746-A750del) EGFR 100 10000NCGC00238624-03 EGFR(G719C) EGFR 100 10000NCGC00238624-03 EGFR(G719S) EGFR 92 10000NCGC00238624-03 EGFR(L747-E749del, A750P) EGFR 72 10000NCGC00238624-03 EGFR(L747-S752del, P753S) EGFR 100 10000NCGC00238624-03 EGFR(L747-T751del,Sins) EGFR 100 10000NCGC00238624-03 EGFR(L858R) EGFR 77 10000NCGC00238624-03 EGFR(L858R,T790M) EGFR 85 10000NCGC00238624-03 EGFR(L861Q) EGFR 100 10000NCGC00238624-03 EGFR(S752-I759del) EGFR 88 10000NCGC00238624-03 EGFR(T790M) EGFR 76 10000NCGC00238624-03 EIF2AK1 EIF2AK1 80 10000NCGC00238624-03 EPHA1 EPHA1 100 10000

S12

NCGC00238624-03 EPHA2 EPHA2 100 10000NCGC00238624-03 EPHA3 EPHA3 94 10000NCGC00238624-03 EPHA4 EPHA4 100 10000NCGC00238624-03 EPHA5 EPHA5 100 10000NCGC00238624-03 EPHA6 EPHA6 100 10000NCGC00238624-03 EPHA7 EPHA7 97 10000NCGC00238624-03 EPHA8 EPHA8 84 10000NCGC00238624-03 EPHB1 EPHB1 100 10000NCGC00238624-03 EPHB2 EPHB2 82 10000NCGC00238624-03 EPHB3 EPHB3 100 10000NCGC00238624-03 EPHB4 EPHB4 100 10000NCGC00238624-03 EPHB6 EPHB6 56 10000NCGC00238624-03 ERBB2 ERBB2 98 10000NCGC00238624-03 ERBB3 ERBB3 94 10000NCGC00238624-03 ERBB4 ERBB4 100 10000NCGC00238624-03 ERK1 MAPK3 100 10000NCGC00238624-03 ERK2 MAPK1 98 10000NCGC00238624-03 ERK3 MAPK6 85 10000NCGC00238624-03 ERK4 MAPK4 89 10000NCGC00238624-03 ERK5 MAPK7 100 10000NCGC00238624-03 ERK8 MAPK15 100 10000NCGC00238624-03 ERN1 ERN1 94 10000NCGC00238624-03 FAK PTK2 100 10000NCGC00238624-03 FER FER 100 10000NCGC00238624-03 FES FES 98 10000NCGC00238624-03 FGFR1 FGFR1 100 10000NCGC00238624-03 FGFR2 FGFR2 100 10000NCGC00238624-03 FGFR3 FGFR3 100 10000NCGC00238624-03 FGFR3(G697C) FGFR3 100 10000NCGC00238624-03 FGFR4 FGFR4 100 10000NCGC00238624-03 FGR FGR 100 10000NCGC00238624-03 FLT1 FLT1 100 10000NCGC00238624-03 FLT3 FLT3 38 10000NCGC00238624-03 FLT3(D835H) FLT3 73 10000NCGC00238624-03 FLT3(D835Y) FLT3 59 10000NCGC00238624-03 FLT3(ITD) FLT3 60 10000NCGC00238624-03 FLT3(K663Q) FLT3 65 10000NCGC00238624-03 FLT3(N841I) FLT3 67 10000NCGC00238624-03 FLT3(R834Q) FLT3 100 10000NCGC00238624-03 FLT4 FLT4 92 10000NCGC00238624-03 FRK FRK 100 10000NCGC00238624-03 FYN FYN 100 10000NCGC00238624-03 GAK GAK 93 10000NCGC00238624-03 GCN2(Kin.Dom.2,S808G) EIF2AK4 100 10000

S13

NCGC00238624-03 GRK1 GRK1 65 10000NCGC00238624-03 GRK4 GRK4 100 10000NCGC00238624-03 GRK7 GRK7 91 10000NCGC00238624-03 GSK3A GSK3A 100 10000NCGC00238624-03 GSK3B GSK3B 88 10000NCGC00238624-03 HCK HCK 100 10000NCGC00238624-03 HIPK1 HIPK1 100 10000NCGC00238624-03 HIPK2 HIPK2 100 10000NCGC00238624-03 HIPK3 HIPK3 88 10000NCGC00238624-03 HIPK4 HIPK4 100 10000NCGC00238624-03 HPK1 MAP4K1 100 10000NCGC00238624-03 HUNK HUNK 86 10000NCGC00238624-03 ICK ICK 93 10000NCGC00238624-03 IGF1R IGF1R 100 10000NCGC00238624-03 IKK-alpha CHUK 100 10000NCGC00238624-03 IKK-beta IKBKB 100 10000NCGC00238624-03 IKK-epsilon IKBKE 84 10000NCGC00238624-03 INSR INSR 93 10000NCGC00238624-03 INSRR INSRR 99 10000NCGC00238624-03 IRAK1 IRAK1 100 10000NCGC00238624-03 IRAK3 IRAK3 95 10000NCGC00238624-03 IRAK4 IRAK4 75 10000NCGC00238624-03 ITK ITK 100 10000NCGC00238624-03 JAK1(JH1domain-catalytic) JAK1 100 10000

NCGC00238624-03JAK1(JH2domain-pseudokinase) JAK1 91 10000

NCGC00238624-03 JAK2(JH1domain-catalytic) JAK2 100 10000NCGC00238624-03 JAK3(JH1domain-catalytic) JAK3 93 10000NCGC00238624-03 JNK1 MAPK8 100 10000NCGC00238624-03 JNK2 MAPK9 89 10000NCGC00238624-03 JNK3 MAPK10 100 10000NCGC00238624-03 KIT KIT 82 10000NCGC00238624-03 KIT(A829P) KIT 83 10000NCGC00238624-03 KIT(D816H) KIT 100 10000NCGC00238624-03 KIT(D816V) KIT 100 10000NCGC00238624-03 KIT(L576P) KIT 63 10000NCGC00238624-03 KIT(V559D) KIT 84 10000NCGC00238624-03 KIT(V559D,T670I) KIT 100 10000NCGC00238624-03 KIT(V559D,V654A) KIT 76 10000NCGC00238624-03 LATS1 LATS1 100 10000NCGC00238624-03 LATS2 LATS2 65 10000NCGC00238624-03 LCK LCK 100 10000NCGC00238624-03 LIMK1 LIMK1 100 10000NCGC00238624-03 LIMK2 LIMK2 100 10000NCGC00238624-03 LKB1 STK11 100 10000

S14

NCGC00238624-03 LOK STK10 90 10000NCGC00238624-03 LRRK2 LRRK2 73 10000NCGC00238624-03 LRRK2(G2019S) LRRK2 100 10000NCGC00238624-03 LTK LTK 100 10000NCGC00238624-03 LYN LYN 92 10000NCGC00238624-03 LZK MAP3K13 67 10000NCGC00238624-03 MAK MAK 100 10000NCGC00238624-03 MAP3K1 MAP3K1 93 10000NCGC00238624-03 MAP3K15 MAP3K15 95 10000NCGC00238624-03 MAP3K2 MAP3K2 46 10000NCGC00238624-03 MAP3K3 MAP3K3 92 10000NCGC00238624-03 MAP3K4 MAP3K4 100 10000NCGC00238624-03 MAP4K2 MAP4K2 100 10000NCGC00238624-03 MAP4K3 MAP4K3 95 10000NCGC00238624-03 MAP4K4 MAP4K4 100 10000NCGC00238624-03 MAP4K5 MAP4K5 100 10000NCGC00238624-03 MAPKAPK2 MAPKAPK2 87 10000NCGC00238624-03 MAPKAPK5 MAPKAPK5 100 10000NCGC00238624-03 MARK1 MARK1 92 10000NCGC00238624-03 MARK2 MARK2 100 10000NCGC00238624-03 MARK3 MARK3 100 10000NCGC00238624-03 MARK4 MARK4 93 10000NCGC00238624-03 MAST1 MAST1 100 10000NCGC00238624-03 MEK1 MAP2K1 87 10000NCGC00238624-03 MEK2 MAP2K2 94 10000NCGC00238624-03 MEK3 MAP2K3 100 10000NCGC00238624-03 MEK4 MAP2K4 100 10000NCGC00238624-03 MEK5 MAP2K5 88 10000NCGC00238624-03 MEK6 MAP2K6 100 10000NCGC00238624-03 MELK MELK 100 10000NCGC00238624-03 MERTK MERTK 84 10000NCGC00238624-03 MET MET 100 10000NCGC00238624-03 MET(M1250T) MET 74 10000NCGC00238624-03 MET(Y1235D) MET 83 10000NCGC00238624-03 MINK MINK1 100 10000NCGC00238624-03 MKK7 MAP2K7 97 10000NCGC00238624-03 MKNK1 MKNK1 98 10000NCGC00238624-03 MKNK2 MKNK2 66 10000NCGC00238624-03 MLCK MYLK3 98 10000NCGC00238624-03 MLK1 MAP3K9 100 10000NCGC00238624-03 MLK2 MAP3K10 100 10000NCGC00238624-03 MLK3 MAP3K11 100 10000NCGC00238624-03 MRCKA CDC42BPA 100 10000NCGC00238624-03 MRCKB CDC42BPB 100 10000

S15

NCGC00238624-03 MST1 STK4 94 10000NCGC00238624-03 MST1R MST1R 100 10000NCGC00238624-03 MST2 STK3 100 10000NCGC00238624-03 MST3 STK24 100 10000NCGC00238624-03 MST4 MST4 52 10000NCGC00238624-03 MTOR FRAP1 100 10000NCGC00238624-03 MUSK MUSK 76 10000NCGC00238624-03 MYLK MYLK 62 10000NCGC00238624-03 MYLK2 MYLK2 90 10000NCGC00238624-03 MYLK4 MYLK4 100 10000NCGC00238624-03 MYO3A MYO3A 100 10000NCGC00238624-03 MYO3B MYO3B 88 10000NCGC00238624-03 NDR1 STK38 92 10000NCGC00238624-03 NDR2 STK38L 100 10000NCGC00238624-03 NEK1 NEK1 96 10000NCGC00238624-03 NEK11 NEK11 100 10000NCGC00238624-03 NEK2 NEK2 100 10000NCGC00238624-03 NEK3 NEK3 89 10000NCGC00238624-03 NEK4 NEK4 100 10000NCGC00238624-03 NEK5 NEK5 67 10000NCGC00238624-03 NEK6 NEK6 100 10000NCGC00238624-03 NEK7 NEK7 100 10000NCGC00238624-03 NEK9 NEK9 100 10000NCGC00238624-03 NIM1 MGC42105 100 10000NCGC00238624-03 NLK NLK 100 10000NCGC00238624-03 OSR1 OXSR1 100 10000NCGC00238624-03 p38-alpha MAPK14 100 10000NCGC00238624-03 p38-beta MAPK11 100 10000NCGC00238624-03 p38-delta MAPK13 100 10000NCGC00238624-03 p38-gamma MAPK12 100 10000NCGC00238624-03 PAK1 PAK1 100 10000NCGC00238624-03 PAK2 PAK2 88 10000NCGC00238624-03 PAK3 PAK3 87 10000NCGC00238624-03 PAK4 PAK4 100 10000NCGC00238624-03 PAK6 PAK6 100 10000NCGC00238624-03 PAK7 PAK7 100 10000NCGC00238624-03 PCTK1 PCTK1 100 10000NCGC00238624-03 PCTK2 PCTK2 87 10000NCGC00238624-03 PCTK3 PCTK3 97 10000NCGC00238624-03 PDGFRA PDGFRA 62 10000NCGC00238624-03 PDGFRB PDGFRB 80 10000NCGC00238624-03 PDPK1 PDPK1 100 10000NCGC00238624-03 PFCDPK1(P.falciparum) PFB0815w 85 10000NCGC00238624-03 PFPK5(P.falciparum) MAL13P1.279 100 10000

S16

NCGC00238624-03 PFTAIRE2 PFTK2 100 10000NCGC00238624-03 PFTK1 PFTK1 100 10000NCGC00238624-03 PHKG1 PHKG1 97 10000NCGC00238624-03 PHKG2 PHKG2 100 10000NCGC00238624-03 PIK3C2B PIK3C2B 100 10000NCGC00238624-03 PIK3C2G PIK3C2G 67 10000NCGC00238624-03 PIK3CA PIK3CA 74 10000NCGC00238624-03 PIK3CA(C420R) PIK3CA 100 10000NCGC00238624-03 PIK3CA(E542K) PIK3CA 100 10000NCGC00238624-03 PIK3CA(E545A) PIK3CA 76 10000NCGC00238624-03 PIK3CA(E545K) PIK3CA 100 10000NCGC00238624-03 PIK3CA(H1047L) PIK3CA 95 10000NCGC00238624-03 PIK3CA(H1047Y) PIK3CA 66 10000NCGC00238624-03 PIK3CA(I800L) PIK3CA 65 10000NCGC00238624-03 PIK3CA(M1043I) PIK3CA 86 10000NCGC00238624-03 PIK3CA(Q546K) PIK3CA 66 10000NCGC00238624-03 PIK3CD PIK3CD 90 10000NCGC00238624-03 PIK3CG PIK3CG 88 10000NCGC00238624-03 PIK4CB PI4KB 66 10000NCGC00238624-03 PIM1 PIM1 99 10000NCGC00238624-03 PIM2 PIM2 86 10000NCGC00238624-03 PIM3 PIM3 94 10000NCGC00238624-03 PIP5K1A PIP5K1A 100 10000NCGC00238624-03 PIP5K1C PIP5K1C 98 10000NCGC00238624-03 PIP5K2B PIP4K2B 100 10000NCGC00238624-03 PIP5K2C PIP4K2C 100 10000NCGC00238624-03 PKAC-alpha PRKACA 100 10000NCGC00238624-03 PKAC-beta PRKACB 100 10000NCGC00238624-03 PKMYT1 PKMYT1 100 10000NCGC00238624-03 PKN1 PKN1 100 10000NCGC00238624-03 PKN2 PKN2 100 10000NCGC00238624-03 PKNB(M.tuberculosis) pknB 57 10000NCGC00238624-03 PLK1 PLK1 100 10000NCGC00238624-03 PLK2 PLK2 100 10000NCGC00238624-03 PLK3 PLK3 100 10000NCGC00238624-03 PLK4 PLK4 66 10000NCGC00238624-03 PRKCD PRKCD 100 10000NCGC00238624-03 PRKCE PRKCE 81 10000NCGC00238624-03 PRKCH PRKCH 100 10000NCGC00238624-03 PRKCI PRKCI 99 10000NCGC00238624-03 PRKCQ PRKCQ 100 10000NCGC00238624-03 PRKD1 PRKD1 100 10000NCGC00238624-03 PRKD2 PRKD2 100 10000NCGC00238624-03 PRKD3 PRKD3 99 10000

S17

NCGC00238624-03 PRKG1 PRKG1 100 10000NCGC00238624-03 PRKG2 PRKG2 97 10000NCGC00238624-03 PRKR EIF2AK2 100 10000NCGC00238624-03 PRKX PRKX 100 10000NCGC00238624-03 PRP4 PRPF4B 100 10000NCGC00238624-03 PYK2 PTK2B 100 10000NCGC00238624-03 QSK KIAA0999 89 10000NCGC00238624-03 RAF1 RAF1 100 10000NCGC00238624-03 RET RET 100 10000NCGC00238624-03 RET(M918T) RET 92 10000NCGC00238624-03 RET(V804L) RET 97 10000NCGC00238624-03 RET(V804M) RET 100 10000NCGC00238624-03 RIOK1 RIOK1 100 10000NCGC00238624-03 RIOK2 RIOK2 93 10000NCGC00238624-03 RIOK3 RIOK3 100 10000NCGC00238624-03 RIPK1 RIPK1 99 10000NCGC00238624-03 RIPK2 RIPK2 100 10000NCGC00238624-03 RIPK4 RIPK4 78 10000NCGC00238624-03 RIPK5 DSTKY 70 10000NCGC00238624-03 ROCK1 ROCK1 90 10000NCGC00238624-03 ROCK2 ROCK2 62 10000NCGC00238624-03 ROS1 ROS1 100 10000

NCGC00238624-03RPS6KA4(Kin.Dom.1-N-terminal) RPS6KA4 98 10000

NCGC00238624-03RPS6KA4(Kin.Dom.2-C-terminal) RPS6KA4 100 10000

NCGC00238624-03RPS6KA5(Kin.Dom.1-N-terminal) RPS6KA5 90 10000

NCGC00238624-03RPS6KA5(Kin.Dom.2-C-terminal) RPS6KA5 100 10000

NCGC00238624-03 RSK1(Kin.Dom.1-N-terminal) RPS6KA1 83 10000NCGC00238624-03 RSK1(Kin.Dom.2-C-terminal) RPS6KA1 100 10000NCGC00238624-03 RSK2(Kin.Dom.1-N-terminal) RPS6KA3 52 10000NCGC00238624-03 RSK3(Kin.Dom.1-N-terminal) RPS6KA2 100 10000NCGC00238624-03 RSK3(Kin.Dom.2-C-terminal) RPS6KA2 100 10000NCGC00238624-03 RSK4(Kin.Dom.1-N-terminal) RPS6KA6 100 10000NCGC00238624-03 RSK4(Kin.Dom.2-C-terminal) RPS6KA6 98 10000NCGC00238624-03 S6K1 RPS6KB1 91 10000NCGC00238624-03 SBK1 SBK1 96 10000NCGC00238624-03 SgK110 SgK110 100 10000NCGC00238624-03 SGK3 SGK3 78 10000NCGC00238624-03 SIK SIK1 100 10000NCGC00238624-03 SIK2 SIK2 100 10000NCGC00238624-03 SLK SLK 100 10000NCGC00238624-03 SNARK NUAK2 99 10000

S18

NCGC00238624-03 SNRK SNRK 89 10000NCGC00238624-03 SRC SRC 100 10000NCGC00238624-03 SRMS SRMS 100 10000NCGC00238624-03 SRPK1 SRPK1 100 10000NCGC00238624-03 SRPK2 SRPK2 100 10000NCGC00238624-03 SRPK3 SRPK3 95 10000NCGC00238624-03 STK16 STK16 100 10000NCGC00238624-03 STK33 STK33 98 10000NCGC00238624-03 STK35 STK35 100 10000NCGC00238624-03 STK36 STK36 100 10000NCGC00238624-03 STK39 STK39 100 10000NCGC00238624-03 SYK SYK 100 10000NCGC00238624-03 TAK1 MAP3K7 98 10000NCGC00238624-03 TAOK1 TAOK1 100 10000NCGC00238624-03 TAOK2 TAOK2 80 10000NCGC00238624-03 TAOK3 TAOK3 93 10000NCGC00238624-03 TBK1 TBK1 100 10000NCGC00238624-03 TEC TEC 100 10000NCGC00238624-03 TESK1 TESK1 100 10000NCGC00238624-03 TGFBR1 TGFBR1 100 10000NCGC00238624-03 TGFBR2 TGFBR2 100 10000NCGC00238624-03 TIE1 TIE1 90 10000NCGC00238624-03 TIE2 TEK 100 10000NCGC00238624-03 TLK1 TLK1 100 10000NCGC00238624-03 TLK2 TLK2 100 10000NCGC00238624-03 TNIK TNIK 100 10000NCGC00238624-03 TNK1 TNK1 100 10000NCGC00238624-03 TNK2 TNK2 100 10000NCGC00238624-03 TNNI3K TNNI3K 100 10000NCGC00238624-03 TRKA NTRK1 84 10000NCGC00238624-03 TRKB NTRK2 65 10000NCGC00238624-03 TRKC NTRK3 46 10000NCGC00238624-03 TRPM6 TRPM6 100 10000NCGC00238624-03 TSSK1B TSSK1B 100 10000NCGC00238624-03 TTK TTK 100 10000NCGC00238624-03 TXK TXK 100 10000NCGC00238624-03 TYK2(JH1domain-catalytic) TYK2 86 10000

NCGC00238624-03TYK2(JH2domain-pseudokinase) TYK2 100 10000

NCGC00238624-03 TYRO3 TYRO3 100 10000NCGC00238624-03 ULK1 ULK1 93 10000NCGC00238624-03 ULK2 ULK2 90 10000NCGC00238624-03 ULK3 ULK3 86 10000NCGC00238624-03 VEGFR2 KDR 100 10000NCGC00238624-03 VRK2 VRK2 100 10000

S19

NCGC00238624-03 WEE1 WEE1 100 10000NCGC00238624-03 WEE2 WEE2 100 10000NCGC00238624-03 YANK1 STK32A 100 10000NCGC00238624-03 YANK2 STK32B 100 10000NCGC00238624-03 YANK3 STK32C 100 10000NCGC00238624-03 YES YES1 100 10000NCGC00238624-03 YSK1 STK25 100 10000NCGC00238624-03 YSK4 YSK4 100 10000NCGC00238624-03 ZAK ZAK 100 10000NCGC00238624-03 ZAP70 ZAP70 28 10000

Experimental Summary

The single crystal X-ray diffraction studies were carried out on a Bruker Kappa APEXII

CCD diffractometer equipped with Mo K radiation ( = 0.71073). A 0.36 x 0.32 x 0.29 mm

colorless plate was mounted on a Cryoloop with Paratone oil. Data were collected in a nitrogen

gas stream at 100(2) K using and scans. Crystal-to-detector distance was 40 mm and

exposure time was 5 s/frame using a scan width of 1.00°. Data collection was 99.9% complete

to 25.41° in . A total of 16130 reflections were collected covering the indices, -30 < h <30, -12

< k < 15, -16 < l < 16. 4164 reflections were found to be symmetry independent, with a R int of

S20

0.0461. Indexing and unit cell refinement indicated a C-centered, monoclinic lattice. The space

group was found to be C2/c. The data were integrated using the Bruker SAINT software

program and scaled using the SADABS software program. Solution by direct methods

(SHELXS) produced a complete phasing model consistent with the proposed structure.

All nonhydrogen atoms were refined anisotropically by full-matrix least-squares

(SHELXL-97). All hydrogen atoms were placed using a riding model. Their positions were

constrained relative to their parent atom using the appropriate HFIX command in SHELXL-97.

Crystallographic data are summarized in Table 1. The TFA anion was located near an inversion

center and was fully disordered. Modeling the disorder proved difficult and the program

SQUEEZE was used to render the anion as a diffuse contribution without specific atom

positions. The intensive properties, however, fully reflect the contribution of the TFA. Found:

400 e-/unit cell. Calc’d for 8 TFA-: 440 e-/unit cell.

S21

S22

Table 1. Crystal data and structure refinement for nih04.

Identification code nih04

Empirical formula C23 H17 F6 N5 O4

Formula weight 541.42

Temperature 100(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group C2/c

Unit cell dimensions a = 25.2479(13) Å = 90°

b = 13.2446(4) Å = 96.880(3)°

c = 13.7031(5) Å = 90°

Volume 4549.3(3) Å3

Z 8

Density (calculated) 1.581 g/cm3

Absorption coefficient 0.143 mm-1

F(000) 2208

Crystal size 0.36 x 0.32 x 0.29 mm3

Theta range for data collection 1.74 to 25.41°

Index ranges -30<=h<=30, -12<=k<=15, -16<=l<=16

Reflections collected 16130

Independent reflections 4164 [R(int) = 0.0461]

Completeness to theta = 25.00° 99.9 %

Absorption correction Multi-scan

Max. and min. transmission 0.9598 and 0.9504

S23

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 4164 / 0 / 279

Goodness-of-fit on F2 1.059

Final R indices [I>2sigma(I)] R1 = 0.0460, wR2 = 0.1208

R indices (all data) R1 = 0.0633, wR2 = 0.1280

Largest diff. peak and hole 0.286 and -0.648 e Å-3

SQUEEZE Found: 400 e/uc. Calc: 8 TFA -, 440e/uc

S24

Table 2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103)for nih04.

U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.

________________________________________________________________________________

x y z U(eq)

________________________________________________________________________________

F(2) 3094(1) 5578(1) 11480(1) 26(1)

F(3) 3253(1) 4150(1) 12195(1) 35(1)

F(1) 2441(1) 4649(1) 11828(1) 31(1)

O(1) 3832(1) 8334(1) 8835(1) 22(1)

O(2) 1393(1) 4410(1) 9867(1) 27(1)

N(1) 3651(1) 7130(1) 9918(1) 17(1)

N(2) 2993(1) 7669(1) 8587(1) 15(1)

N(3) 2683(1) 6349(1) 9530(1) 15(1)

N(4) 2167(1) 6998(1) 8161(1) 15(1)

N(5) 2852(1) 3115(1) 8721(1) 17(1)

C(1) 4180(1) 7448(1) 10146(1) 18(1)

C(2) 4288(1) 8189(1) 9485(2) 20(1)

C(3) 4772(1) 8662(2) 9505(2) 28(1)

C(4) 5169(1) 8357(2) 10234(2) 28(1)

C(5) 5070(1) 7609(2) 10906(2) 25(1)

C(6) 4576(1) 7140(2) 10875(2) 23(1)

C(7) 3469(1) 7663(1) 9139(1) 15(1)

C(8) 2632(1) 6999(1) 8782(1) 13(1)

S25

C(9) 2307(1) 5541(1) 9709(1) 13(1)

C(10) 2589(1) 3985(1) 8809(1) 14(1)

C(11) 2594(1) 4527(1) 9686(1) 12(1)

C(12) 2895(1) 4111(1) 10515(1) 16(1)

C(13) 3164(1) 3207(1) 10436(1) 19(1)

C(14) 3132(1) 2737(1) 9531(1) 19(1)

C(15) 2923(1) 4619(2) 11501(1) 22(1)

C(16) 1763(1) 6324(1) 8258(1) 15(1)

C(17) 1272(1) 6441(2) 7544(2) 20(1)

C(18) 950(1) 5478(2) 7443(2) 29(1)

C(19) 864(1) 5042(2) 8430(2) 28(1)

C(20) 1366(1) 4981(1) 9147(2) 20(1)

C(21) 1809(1) 5618(1) 8984(1) 15(1)

Table 3. Bond lengths [Å] and angles [°] for nih04.

_____________________________________________________

F(2)-C(15) 1.343(2)

F(3)-C(15) 1.339(2)

F(1)-C(15) 1.348(3)

O(1)-C(7) 1.377(2)

O(1)-C(2) 1.381(2)

O(2)-C(20) 1.238(2)

N(1)-C(7) 1.315(2)

N(1)-C(1) 1.400(3)

N(2)-C(8) 1.321(2)

N(2)-C(7) 1.341(2)

N(3)-C(8) 1.333(2)

N(3)-C(9) 1.470(2)

N(4)-C(8) 1.365(2)

N(4)-C(16) 1.375(2)

N(5)-C(14) 1.339(2)

N(5)-C(10) 1.343(2)

S26

C(1)-C(2) 1.386(3)

C(1)-C(6) 1.388(3)

C(2)-C(3) 1.371(3)

C(3)-C(4) 1.389(3)

C(4)-C(5) 1.395(3)

C(5)-C(6) 1.389(3)

C(9)-C(21) 1.508(3)

C(9)-C(11) 1.529(3)

C(10)-C(11) 1.399(3)

C(11)-C(12) 1.401(3)

C(12)-C(13) 1.388(3)

C(12)-C(15) 1.504(3)

C(13)-C(14) 1.381(3)

C(16)-C(21) 1.360(3)

C(16)-C(17) 1.493(3)

C(17)-C(18) 1.510(3)

C(18)-C(19) 1.511(3)

C(19)-C(20) 1.509(3)

C(20)-C(21) 1.441(3)

C(7)-O(1)-C(2) 104.47(14)

C(7)-N(1)-C(1) 104.56(16)

C(8)-N(2)-C(7) 118.32(16)

C(8)-N(3)-C(9) 126.50(17)

C(8)-N(4)-C(16) 122.21(16)

C(14)-N(5)-C(10) 117.67(16)

C(2)-C(1)-C(6) 119.86(19)

C(2)-C(1)-N(1) 108.89(17)

C(6)-C(1)-N(1) 131.25(18)

C(3)-C(2)-O(1) 128.45(19)

C(3)-C(2)-C(1) 123.73(19)

O(1)-C(2)-C(1) 107.81(17)

C(2)-C(3)-C(4) 116.6(2)

C(3)-C(4)-C(5) 120.7(2)

C(6)-C(5)-C(4) 121.9(2)

C(5)-C(6)-C(1) 117.3(2)

N(1)-C(7)-N(2) 131.55(18)

N(1)-C(7)-O(1) 114.26(17)

N(2)-C(7)-O(1) 114.19(16)

N(2)-C(8)-N(3) 125.80(17)

N(2)-C(8)-N(4) 115.88(16)

N(3)-C(8)-N(4) 118.31(17)

N(3)-C(9)-C(21) 110.11(15)

N(3)-C(9)-C(11) 108.55(15)

S27

C(21)-C(9)-C(11) 113.97(15)

N(5)-C(10)-C(11) 124.23(17)

C(10)-C(11)-C(12) 116.38(16)

C(10)-C(11)-C(9) 120.79(16)

C(12)-C(11)-C(9) 122.77(16)

C(13)-C(12)-C(11) 119.93(17)

C(13)-C(12)-C(15) 119.00(17)

C(11)-C(12)-C(15) 121.06(17)

C(14)-C(13)-C(12) 118.79(17)

N(5)-C(14)-C(13) 123.00(18)

F(3)-C(15)-F(2) 106.33(16)

F(3)-C(15)-F(1) 106.69(16)

F(2)-C(15)-F(1) 106.53(16)

F(3)-C(15)-C(12) 112.92(17)

F(2)-C(15)-C(12) 112.66(16)

F(1)-C(15)-C(12) 111.27(17)

C(21)-C(16)-N(4) 121.21(18)

C(21)-C(16)-C(17) 122.90(18)

N(4)-C(16)-C(17) 115.86(16)

C(16)-C(17)-C(18) 111.56(16)

C(17)-C(18)-C(19) 111.98(18)

C(20)-C(19)-C(18) 113.83(18)

O(2)-C(20)-C(21) 120.70(18)

O(2)-C(20)-C(19) 120.94(18)

C(21)-C(20)-C(19) 118.36(17)

C(16)-C(21)-C(20) 121.04(18)

C(16)-C(21)-C(9) 121.33(17)

C(20)-C(21)-C(9) 117.37(16)

S28

_____________________________________________________

S29

Table 4. Anisotropic displacement parameters (Å2x 103) for nih04. The anisotropic displacement factor

exponent takes the form: -22[ h2 a*2U11 + ... + 2 h k a* b* U12]

______________________________________________________________________________

U11 U22 U33 U23 U13 U12

______________________________________________________________________________

F(2) 40(1) 18(1) 20(1) -2(1) -1(1) -7(1)

F(3) 58(1) 29(1) 15(1) 4(1) -10(1) 5(1)

F(1) 47(1) 30(1) 19(1) -1(1) 17(1) -3(1)

O(1) 17(1) 22(1) 26(1) 8(1) 0(1) -6(1)

O(2) 19(1) 24(1) 38(1) 17(1) 5(1) -2(1)

N(1) 16(1) 14(1) 19(1) 1(1) 1(1) -1(1)

N(2) 18(1) 13(1) 15(1) 6(1) 2(1) -1(1)

N(3) 14(1) 13(1) 17(1) 2(1) 0(1) -1(1)

N(4) 16(1) 14(1) 17(1) 6(1) 3(1) -2(1)

N(5) 18(1) 14(1) 20(1) 0(1) 5(1) 1(1)

C(1) 18(1) 15(1) 21(1) -3(1) 3(1) -3(1)

C(2) 18(1) 19(1) 22(1) 1(1) 0(1) -2(1)

C(3) 23(1) 27(1) 33(1) 6(1) 3(1) -10(1)

C(4) 18(1) 31(1) 34(1) -4(1) 4(1) -8(1)

C(5) 18(1) 27(1) 28(1) -2(1) -2(1) 0(1)

C(6) 21(1) 21(1) 25(1) 0(1) 0(1) 0(1)

C(7) 18(1) 9(1) 19(1) -1(1) 6(1) -4(1)

C(9) 16(1) 11(1) 15(1) 0(1) 5(1) -1(1)

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C(10) 15(1) 13(1) 14(1) 1(1) 4(1) -1(1)

C(11) 12(1) 12(1) 13(1) 2(1) 4(1) -3(1)

C(12) 19(1) 15(1) 15(1) 3(1) 3(1) -4(1)

C(13) 21(1) 17(1) 19(1) 5(1) 0(1) 3(1)

C(14) 22(1) 12(1) 23(1) 2(1) 5(1) 3(1)

C(15) 34(1) 17(1) 15(1) 2(1) 1(1) -3(1)

C(16) 14(1) 11(1) 19(1) -4(1) 4(1) 2(1)

C(17) 14(1) 25(1) 21(1) 6(1) 2(1) 0(1)

C(18) 26(1) 27(1) 32(1) 1(1) -6(1) -6(1)

C(19) 20(1) 24(1) 39(1) 9(1) -1(1) -7(1)

C(20) 17(1) 15(1) 28(1) 3(1) 5(1) 1(1)

C(21) 15(1) 13(1) 18(1) -1(1) 6(1) 2(1)

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Table 5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2 x 10 3) for nih04.

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x y z U(eq)

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H(2A) 2923 8107 8106 18

H(4B) 2125 7443 7681 18

H(3A) 4832 9173 9044 33

H(4A) 5512 8660 10276 33

H(5A) 5349 7414 11398 30

H(6A) 4513 6630 11334 27

H(9A) 2202 5637 10384 16

H(10A) 2385 4249 8238 16

H(13A) 3368 2917 10995 23

H(14A) 3316 2117 9481 22

H(17A) 1374 6633 6894 24

H(17B) 1049 6991 7767 24

H(18A) 1138 4976 7074 35

H(18B) 599 5617 7061 35

H(19A) 600 5464 8722 34

H(19B) 712 4356 8330 34

H(3) 2987(10) 6415(16) 9907(17) 29(6)

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