S1

Supporting Information

Discovery of a Diaminopyrimidine FLT3 Inhibitor Active Against

Acute Myeloid Leukemia

Jamie A. Jarusiewicz†, Jae Yoon Jeon

§,, Michele C. Connelly

†, Yizhe Chen

†,‡, Lei Yang

†, Sharyn D.

Baker§, and R. Kiplin Guy

*†,‡

†Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas

Place, Memphis, Tennessee 38105, United States

§Division of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 W. 12th St., Columbus, Ohio,

43210, United States

‡Current affiliation: Department of Pharmaceutical Sciences, University of Kentucky College of Pharmacy, 214H

BioPharm Complex, Lexington, Kentucky, 40536, United States

Table of Contents

Experimental procedures for ADME and PK studies S2-S5

Additional synthetic schemes Schemes S1-S6

Additional synthetic procedures and compound characterization S7-S9

KinomeScan analysis for compound 1 Table S1

FLT3 and MV4-11 data with confidence intervals Table S2

BJ growth inhibition data Table S3

Solubility and PAMPA permeability Table S4

PGP Efflux Assay Table S5

In vivo blood chemistry data after i.p. dosing of 5e Table S6

S2

Experimental procedures for ADME and PK studies.

Solubility. Solubility assays were carried out on a Biomek FX lab automation workstation

(Beckman Coulter, Inc., Fullerton, CA) using µSOL Evolution software (pION Inc., Woburn,

MA). The detailed method is described as follows. 10 µL of 10 mM compound stock (in DMSO)

was added to 190 µL 1-propanol to make a reference stock plate. 5 µL from this reference stock

plate were mixed with 70 µL 1-propanol and 75 µL citrate phosphate-buffered saline (PBS;

isotonic) to make the reference plate, and the UV spectrum (250-500 nm) of the reference plate

was read. 6 µL of 10 mM test compound stock was added to 594 µL buffer in a 96-well storage

plate and mixed. The storage plate was sealed and incubated at RT for 18 h. The suspension was

then filtered through a 96-well filter plate (pION Inc., Woburn, MA). 75 µL of filtrate was mixed

with 75 µL 1-propanol to make the sample plate, and the UV spectrum of the sample plate was

read. Calculations were carried out with µSOL Evolution software based on the area under the

curve (AUC) of the UV spectrum of the sample and reference plates. All compounds were tested

in triplicate.

Permeability. Our parallel artificial membrane permeability assay (PAMPA) was conducted

with a Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) and

PAMPA Evolution 96 Command software (pION Inc., Woburn, MA). The detailed method is

described as follows: 3 µL of 10 µM test compound stock in DMSO was mixed with 597 µL of

citrate PBS (isotonic) to make a diluted test compound. 150 µL of diluted test compound was

transferred to a UV plate (pION Inc., Woburn, MA), and the UV spectrum of this reference plate

was read. The membrane, on a pre-loaded PAMPA Sandwich (pION Inc., Woburn, MA), was

painted with 4 µL of GIT lipid (pION Inc., Woburn, MA). The acceptor chamber was then filled

with 200 µL of ASB (acceptor solution buffer, pION Inc., Woburn, MA), and the donor chamber

was filled with 180 µL of diluted test compound. The PAMPA Sandwich was assembled, placed

on the Gut-box, and stirred for 30 min. The aqueous boundary layer was set to 40 µm for stirring.

The UV spectrum (250-500 nm) of the donor and the acceptor were read. The permeability

coefficient was calculated using PAMPA Evolution 96 Command software based on the AUC of

the reference plate, the donor plate, and the acceptor plate. All compounds were tested in

triplicate.

Plasma stability. Compound stocks were at a concentration of 10 mM in DMSO. The internal

standard was 10 µM warfarin in methanol. 1.9 mL of mouse plasma (Fisher Scientific) or pooled

human plasma (Innovative Research Inc) were added to columns 1, 4, 7, and 10 of a 2 mL 96-

well deep well plate (pION Inc., Woburn, MA, catalog #110023); this was the master plate. 1.9

µL of compound stock were added to each well with plasmas and mixed well. Using a multi-

channel pipette, 600 µL from columns 1, 4, 7, and 10 were taken and added into the rest of the

columns (fluids in column 1 were added to columns 2 and 3, column 4 to 5 and 6, etc.). From the

master plate, 65 µL was taken from each well and added into eight storage plates (pION Inc.,

Woburn, MA, catalog #110323), each for a different time point. The storage plates were then

S3

incubated at 37 ºC and shaken at 60 rpm. Samples were taken at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h,

24 h, and 48h. At each time point, 195 µL of internal standard was added to quench the reaction.

The plates were then centrifuged at 4000 rpm for 15 min, and supernatant was analyzed by Ultra

High Pressure Liquid Chromatography with Mass Spectrometry (UPLC-MS; Waters Inc.,

Milford, MA). The compound was detected by selected ion recording (SIR), and quantitation

was based on the peak area ratio of the test compound vs. the internal standard.

Simulated gastric fluid (SGF) stability. Compound stocks were at a concentration of 10 mM in

DMSO. The internal standard was 10 µM warfarin in methanol. 1.4 mL of concentrated HCl

(37%), 0.4 g of NaCl, and 0.64 g of pepsin were added to 198 mL of deionized water to make

SGF (pH 1). 1.9 mL of SGF were added to columns 1, 4, 7, and 10 of a 2 mL 96-well deep well

plate; this was the master plate. 1.9 µL of compound stock was added to each well with SGF and

mixed well. Using a multi-channel pipette, 600 µL from columns 1, 4, 7, and 10 was taken and

added into the rest of the columns (fluids in column 1 were added to columns 2 and 3, column 4

to 5 and 6, etc.). From the master plate, 65 µL was taken from each well and added into eight

storage plates, each for a different time point. The storage plates was then incubated at 37 ºC and

shaken at 60 rpm. Samples were taken at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48h. At each

time point, 195 µL of internal standard was added to quench the reaction. The plates were then

centrifuged at 4000 rpm for 15 min, and supernatant was analyzed by UPLC-MS. The compound

was detected by SIR, and quantitation was based on the peak area ratio of the test compound vs.

the internal standard.

PBS stability. Compound stocks were at a concentration of 10 mM in DMSO. The internal

standard was 10 µM warfarin in methanol. 1.9 mL of PBS (Mediatech Inc., Manassas, VA,

catalog #21-040-CM) were added to columns 1, 4, 7, and 10 of a 2 mL 96-well deep well plate;

this was the master plate. 1.9 µL of compound stock were added to each well with SGF and

mixed well. Using a multi-channel pipette, 600 µL from columns 1, 4, 7, and 10 was taken and

added into the rest of the columns (fluids in column 1 were added to columns 2 and 3, column 4

to 5 and 6, etc.). From the master plate, 65 µL was taken from each well and added into eight

storage plates, each for a different time point. The storage plates were then incubated at 37 ºC

and shaken at 60 rpm. Samples were taken at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. At

each time point, 195 µL of internal standard was added to quench the reaction. The plates were

then centrifuged at 4000 rpm for 15 min, and supernatant was analyzed by UPLC-MS. The

compound was detected by SIR, and quantitation was based on the peak area ratio of the test

compound vs. the internal standard.

Liver microsomal stability. 1.582 mL of mouse liver microsome (20 mg/mL, female CD-1

mice, pooled; Fisher Scientific, catalog #NC9567486) were mixed with 0.127 mL of 0.5 M

EDTA solution and 48.3 mL of potassium phosphate buffer (0.1 M, pH 7.4, 37 °C) to make 50

mL of mouse liver microsome solution. Human liver microsomal solution was made in the same

way with human liver microsome (50 pooled mix gender; Fisher Scientific, catalog #50-722-

516). One volume of 10 mM DMSO compound stock was mixed with four volumes of

acetonitrile to make 2 mM diluted compound stock in DMSO and acetonitrile. 37.83 µL of

diluted compound stock was added to 3 mL of liver microsomal solution and vortexed to make

microsomal solution with compound. 1 mL of liver microsomal solution with compound was

added to each well of a master storage plate (pION Inc., Woburn, MA, catalog #110323). All

compounds were added in triplicate. Mouse and human liver microsomes were tested side by

S4

side on the same plate. 175 µL of each well was dispensed from the master plate into five storage

plates. For 0 h time point, 450 µL of pre-cooled (4 ºC) internal standard (10 µM warfarin in

methanol) was added to the first plate before the reaction started. 5.25 mL of microsome assay

solution A (Fisher Scientific, catalog #NC9255727) was combined with 1.05 mL of solution B

(Fisher Scientific, catalog #NC9016235) in 14.7 mL of potassium phosphate buffer (0.1 M, pH

7.4). 45 µL of this A+B solution was added to each well of all the 96-well storage plates and

mixed briefly with pipette. The plates were sealed, and all plates except the 0 h plate were

incubated at 37 ºC, with shaking at 60 rpm. 0.5 h, 1 h, 2 h, and 4 h time points were taken. At

each time point, 450 µL of pre-cooled internal standard was added to quench the reaction. The

quenched plate was then centrifuged (model 5810R, Eppendorf, Westbury, NY) at 4000 rpm for

15 min. 150 µL of supernatant was transferred to a 96-well plate and analyzed by UPLC-MS.

The compounds and internal standard were detected by SIR. The log peak area ratio (compound

peak area / internal standard peak area) was plotted vs. time (h), and the slope was determined to

calculate the elimination rate constant [k = (-2.303) * slope]. The half-life (h) was calculated as t

1/2 = 0.693 / k. Intrinsic clearance was calculated as CLint = (0.693 / (t1/2) * (1 / microsomal

concentration in the reaction solution) * (45 mg microsome / g liver) * (g liver / kg b.w.), where

microsomal concentration in the reaction solution is 0.5 mg/mL, and g liver / kg b.w. of CD-1

female mice and human are 52 and 20, respectively.

Caco-2 and MDR1-MDCKII permeability assay. High throughput Caco-2 and MDR1-

MDCKII permeability was performed in the Transwell® 0.4 µm polycarbonate membrane 96-

well system with modified methods.1, 2

Caco-2 and MDR1-MDCKII cells were maintained at 37

°C in a humidified incubator with an atmosphere of 5% CO2. The cells were cultured in 75 cm2

flasks with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum

(FBS), 1% non-essential amino acids (NEAA), 100 units/ml of penicillin, and 100 µg/ml of

streptomycin. The Caco-2 and MDR1-MDCKII cells were seeded onto inserts at a density of

2×104 and 1×10

4 cells/insert separately. The medium in the wells was exchanged each other day,

and the trans epithelial electrical resistance (TEER) value was measured using an epithelial volt-

ohm meter (Millipore, Billerica, Massachusetts). Caco2 cells were grown for 7 days and MDR1-

MDCKII cells were grown for 4 days to reach consistent TEER values (typically 2000 ohms

greater than initial value when cells are first seeded into transwells), indicating that the cells had

formed a confluent polarized monolayer.

For transport experiments, each cultured monolayer on the 96-well plate was washed twice with

a transport buffer (HBSS/25 mM HEPES, pH 7.4). The permeability assay was initiated by the

addition of each compound solution (10 µmol/L) into inserts (apical side, A) or receivers

(basolateral side, B). The Caco-2 cell monolayers were incubated for 2 h at 37 °C and the

MDR1-MDCKII cell monolayers were incubated for 1 hour at 37 °C. Fractions were collected

from receivers (if apical to basal permeability) or inserts (if basal to apical permeability), and

concentrations were assessed by UPLC/MS (Waters; Milford, MA). All compounds were tested

in triplicates.

The A→B (or B→A) apparent permeability coefficients (Papp) of each compound were

calculated using the equation, Papp=dQ/dt×1/AC0, where dQ/dt equals the flux of a drug across

the monolayer, A equals the total insert well surface area, and C0 is the initial concentration of

substrate in the donor compartment. The efflux ratio was determined by dividing the Papp in the

S5

B-A direction by the Papp in the A-B direction. An efflux ratio >2 suggested that a given substrate

was actively transported across the membrane.

Maximum tolerated dose (MTD) study in mice. During the acute observation phase after

dosing, no adverse reactions or compound-related side effects were observed based on based on

a functional observational battery. All animals maintained normal feeding behavior and body

weight during the 48 h period following i.p. administration of compound at all doses. No

significant changes to hematological parameters, clinical chemistries, or gross organ anatomy

were observed at the terminal point of the study. However, previous pilot study suggested rapid

neurotic/cardiac driven toxicity at 50 mg/kg, and a plunge of plasma glucose levels were

observed within 30 minutes post injection (data not shown). The blood work of compound 5e

after IP dosing is summarized in Table S6.

PK analysis was conducted on an AB Sciex 6500 coupled to Waters Acquity UPLC

(LC/MS/MS) using positive electrospray ionization multiple reaction monitoring (MRM) mode.

An Acquity BEH C18, 1.7 µM, 2.1*50 mm column with acetonitrile (B) – water (A) gradient

containing 0.1% formic acid as mobile phase. LC conditions were a gradient (98-20% A), cycle

time, 2 min. using an injection volume of 2.5 µL and flow rate of 0.6 mL/min. The calibration

curve was obtained as follows: The calibration was done using the same biological matrices to

ensure the similar recovery. Serial dilution (1:2) of compound stock solution (in DMSO) was

spiked into 99 volumes of blank mouse plasma (final concentration from 13.7 to 3333 nM). 1

volume of spiked plasma was combined with 3 volumes of internal standard (2 µM warfarin in

acetonitrile) to precipitate the mouse plasma proteins. all concentrations in the calibration range

must be no more than 20% deviation. If there were any points with more than 20% deviation on

the high or low end, the calibration range would be truncated. If an outlier occurred in the middle

of the curve, the whole curve would be re-done. The signal: noise of the compound peak at the

LLOQ must be greater than 5. Partial validation is achieved if >75% duplicated calibrations,

>2/3rd of total QCs, >50% QC from each level, meet acceptance interval <20% for LLOQ, 15%

for other levels. The samples are tested on the same or second day and another set of QC ran

immediately after sample analysis.

Additional synthetic schemes.

Scheme S1. Preparation of intermediates 24 and 25 via Suzuki Reactionsa

S6

aReagents and conditions: (a) 3-bromo-N-methylaniline, K2CO3, Pd(Ph3)4, dioxane/water (3:1),

80 °C, 16 h, 81%; (b) 3-bromophenol, K2CO3, Pd(Ph3)4, dioxane/water (3:1), 80 °C, 16 h, 70%.

Scheme S2. Preparation of intermediate 29a

aReagents and conditions: (a) K2CO3, Pd(Ph3)4, dioxane/water (3:1), 80 °C, 16 h, 54%; (b)

trifluoroacetic acid, DCM, rt, 3 h, 82%.

Scheme S3. Synthesis of intermediate 32a

aReagents and conditions: (a) 3-pyridinyl pinacol boronic ester, potassium phosphate,

[Rh(OH)(1,5-cod)]2, dioxane/water, 80 °C, 16 h, 75%; (b) i. titanium (IV) isopropoxide,

ammonium chloride, triethylamine, ethanol, rt, 16 h; ii. sodium borohydride, ethanol, rt, 7 h

29%.3, 4

Scheme S4. Preparation of 5i by hydrolysisa

N

NHN

HO

OHN

N

5i

a

N

NHN

H3CO

OHN

N

5h

aReagents and conditions: (a) NaOH, THF/water (5:1), 80 °C, 3 h, 49%.

Scheme S5. Preparation of intermediate 3ma

aReagents and conditions: (a) 1-(trifluoromethyl)cyclopropanamine, NMP, µwave, 140 °C, 1 h,

3%.

Scheme S6. Deprotection of intermediate 3r to prepare 5qa

S7

aReagents and conditions: (a) trifluoroacetic acid, DCM, rt, 3 h, quantitative.

Additional synthetic procedures and compound characterization.

N-Methyl-3-(pyridin-3-yl)aniline (24). A mixture of 3-bromo-N-methylaniline (1.0

mmol, 1.0 eq.), pyridin-3-ylboronic acid (1.2 mmol, 1.2 eq.), potassium carbonate (3.0 mmol, 3.0

eq.), and Pd(Ph3)4 (0.050 mmol, 5 mol%) in 2.0 mL (0.50M) dioxane/water (3:1) was degassed

then stirred in a nitrogen atmosphere at 80 °C for 16 h. After cooling to room temperature the

reaction mixture was partitioned between ethyl acetate (3 mL) and water (2 mL). The aqueous

phase was extracted into ethyl acetate (3 x 3 mL). The combined organics were dried over

magnesium sulfate, filtered, and concentrated. Purification using automated flash

chromatography (EtOAc/hexanes) was followed by evaporation giving 24 as a yellow oil (0.150

g, 81%). TLC Rf 0.50 (80% EtOAc/hexanes). LC-MS (ESI) m/z: 185 [M + H]+.

1H NMR (400

MHz, CDCl3) δ 8.86 (dd, J = 2.3, 0.9 Hz, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 7.89 (dt, J = 7.9,

2.0 Hz, 1H), 7.39 – 7.29 (m, 2H), 6.94 (d, J = 4.0 Hz, 1H), 6.81 (t, J = 2.1 Hz, 1H), 6.68 (ddd, J

= 8.0, 2.4, 1.0 Hz, 1H), 3.77 (br s, 1H), 2.92 (s, 3H). 13

C NMR (101 MHz, CDCl3) δ 149.85,

148.36, 148.30, 138.92, 137.25, 134.42, 129.90, 123.43, 116.21, 112.21, 110.87, 30.73.

3-(Pyridin-3-yl)phenol (25). A mixture of 3-bromophenol (1.0 mmol, 1.0 eq.), pyridin-3-

ylboronic acid (1.1 mmol, 1.1 eq.) potassium carbonate (3.0 mmol, 3.0 eq.), and Pd(Ph3)4 (0.050

mmol, 5 mol%) in 2.0 mL (0.50M) of dioxane/water (3:1) was degassed then stirred at 80 °C in a

nitrogen atmosphere. After 16 h, the reaction mixture was cooled to room temperature and

diluted with water (3 mL). The reaction mixture was then extracted into ethyl acetate (3 x 3 mL).

S8

The combined organics were washed with saturated sodium bicarbonate solution (5mL) followed

by brine (5mL). The organic phase was dried over magnesium sulfate, filtered, and

concentrated. Purification using automated flash chromatography (EtOAc/hexanes) was

followed by evaporation giving 25 as a white solid (0.119 g, 70%). TLC Rf 0.5 (80%

EtOAc/hexanes). LC-MS (ESI) m/z: 172 [M + H]+.

1H NMR (500 MHz, MeOD) δ 8.78 (s, 1H),

8.53 (d, J = 5.0, 1H), 8.08 (dt, J = 8.0, 1.9 Hz, 1H), 7.53 (dd, J = 8.0, 4.9 Hz, 1H), 7.33 (t, J = 7.9

Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.07 (s, 1H), 6.94 – 6.83 (m, 1H). 13

C NMR (126 MHz,

MeOD) δ 157.94, 147.29, 146.87, 138.49, 137.38, 135.10, 129.94, 124.03, 117.83, 115.01,

113.40.

tert-Butyl (3-(pyrazin-2-yl)phenyl)carbamate (28). A mixture of 2-chloropyrazine (0.50

mmol, 1.0 eq.), 3-(N-butoxycarbonyl)aminophenylboronic acid (0.55 mmol, 1.1 eq.), potassium

carbonate (1.5 mmol, 3.0 eq.), and Pd(PPh3)4 (0.025 mmol, 5 mol%) catalyst in 1.0 mL (0.50M)

dioxane/water (3:1) was degassed then stirred in a nitrogen atmosphere at 80 °C for 16 h. After

cooling to room temperature the reaction mixture was diluted with ethyl acetate (3 mL) and

washed sequentially with saturated sodium bicarbonate solution (3 mL) and brine (3 mL). The

organic phase was dried over magnesium sulfate, filtered, and concentrated. Purification using

automated flash chromatography (EtOAc/hexanes) was followed by evaporation giving 28 as a

white solid (0.073 g, 54%). TLC Rf 0.3 (30% EtOAc/hexanes). LC-MS (ESI) m/z: 272 (M +

H)+.

1H NMR (500 MHz, CDCl3) δ 9.05 (br s, 1H), 8.64 (s, 1H), 8.53 (d, J = 2.5 Hz, 1H), 8.11

(s, 1H), 7.69 (d, J = 10.0 Hz, 1H), 7.54 – 7.48 (m, 1H), 7.45 (t, J = 7.8 Hz, 1H), 6.64 (s, 1H),

1.56 (s, 9H). 13

C NMR (126 MHz, CDCl3) δ 152.65, 152.47, 144.13, 143.09, 142.32, 139.24,

137.21, 129.71, 121.44, 119.86, 116.90, 80.82, 28.34.

S9

3-(Pyrazin-2-yl)aniline (29). To 28 (0.26 mmol, 1.0 eq.) in dichloromethane (1.0 mL,

0.26M) in a nitrogen atmosphere at room temperature was added trifluoroacetic acid (2.6 mmol,

10 eq). After stirring at room temperature for 3 h, the reaction mixture was

concentrated.Purification using automated flash chromatography (MeOH/DCM) was followed

by evaporation giving 29 as a white solid (0.036 g, 82%). TLC Rf 0.5 (10% MeOH/DCM). LC-

MS (ESI) m/z: 172 (M + H)+.

1H NMR (400 MHz, MeOD) δ 9.16 (d, J = 1.5 Hz, 1H), 8.73 (dd, J

= 2.5, 1.5 Hz, 1H), 8.61 (d, J = 2.5 Hz, 1H), 8.09 – 7.95 (m, 2H), 7.62 (t, J = 8.0 Hz, 1H), 7.38

(ddd, J = 8.0, 2.3, 1.0 Hz, 1H). 13

C NMR (101 MHz, MeOD) δ 152.92, 145.80, 144.74, 143.18,

139.41, 137.31, 131.77, 125.69, 123.56, 120.59.

3-(Pyridin-3-yl)cyclohexan-1-one (31). A mixture of [Rh(OH)(1,5-cod)]2 (0.025 mmol,

2.5 mol%) was stirred in dioxane (1.4 mL) in a nitrogen atmosphere for 15 min., then potassium

phosphate (4.0 mmol, 4.0 eq.) in water (2.1 mL) was added and stirred for an additional 15 min.

at room temperature. Cyclohexenone (1.0 mmol, 1.0 eq.) and 3-pyridinyl pinacol boronic ester

(2.0 mmol, 2.0 eq.) were added and the reaction mixture (0.29 M) was stirred at 80 °C. After 16

h, the reaction mixture was cooled to room temperature. Saturated sodium bicarbonate (5 mL)

was added to the reaction mixture and extracted into ethyl acetate (3 x 10 mL). The combined

organics were washed with brine (20 mL), then dried the organic phase over magnesium sulfate,

filtered and concentrated. Purification using automated flash chromatography (EtOAc/hexanes)

was followed by evaporation giving 31 as a yellow oil (0.131 g, 75%). TLC Rf 0.25 (80%

EtOAc/hexanes). LC-MS (ESI) m/z: 176 (M + H)+.

1H NMR (500 MHz, CDCl3) δ 8.61 – 8.45

(m, 2H), 7.57 (dt, J = 8.0, 1.9 Hz, 1H), 7.35 – 7.21 (m, 1H), 3.07 (ddd, J = 16.0, 8.0, 4.0 Hz, 1H),

2.63 (ddt, J = 14.0, 4.3, 2.0 Hz, 1H), 2.59 – 2.47 (m, 2H), 2.42 (ddd, J = 14.1, 12.3, 6.2 Hz, 1H),

2.21 (ddd, J = 13.2, 6.3, 3.1 Hz, 1H), 2.16 – 2.09 (m, 1H), 1.97 – 1.75 (m, 2H). 13

C NMR (126

S10

MHz, CDCl3) δ 209.94, 148.56, 148.22, 139.43, 134.00, 123.61, 48.39, 42.24, 41.05, 32.44,

25.40.

3-(Pyridin-3-yl)cyclohexan-1-amine (32). A mixture of 31 (0.28 mmol, 1.0 eq.), titanium

(IV) isopropoxide (0.56 mmol, 2.0 eq.), ammonium chloride (0.56 mmol, 2.0 eq.), and

triethylamine (0.56 mmol, 2.0 eq.) were stirred at room temperature in EtOH (2.0 mL, 0.19M) in

a nitrogen atmosphere for 16 h. Sodium borohydride (0.57 mmol, 1.5 eq.) was then added and

the reaciton mixture was allowed to stir at room temperature for an additional 7 h. The reaction

was then quenched with saturated sodium bicarbonated solution (2 mL) and extracted into ethyl

acetate (3 x 5 mL). A thick paste formed and the ethyl acetate was decanted, then washed with

brine, dried organic phase over magnesium sulfate, filtered and concentrated. Purification using

automated flash chromatography (MeOH/DCM) was followed by evaporation giving 32 as a

yellow oil (0.019 g, 29%). TLC Rf 0.4 (10% MeOH/DCM). LC-MS (ESI) m/z: 178 (M + H)+.

1H NMR (400 MHz, CDCl3) δ 8.61 – 8.40 (m, 2H), 7.57 (dt, J = 8.0, 2.0 Hz, 1H), 7.27 (d, J =

4.6 Hz, 1H), 3.87 – 3.70 (m, 1H), 2.73 – 2.56 (m, 1H), 2.26 – 2.15 (m, 1H), 2.11 (d, J = 12.3 Hz,

1H), 2.01 – 1.91 (m, 1H), 1.91 – 1.82 (m, 1H), 1.55 – 1.41 (m, 2H), 1.40 – 1.24 (m, 2H). 13

C

NMR (101 MHz, CDCl3) δ 148.68, 147.50, 141.28, 134.23, 123.51, 70.68, 42.73, 40.28, 35.21,

33.12, 24.34.

Table S1. KinomeScan analysis for compound 1

Kinase

Cmpd 1

(10 µM)

% Controla

AAK1 33

ABL1(E255K)-

phosphorylated 14

ABL1(F317I)- 100

S11

nonphosphorylated

ABL1(F317I)-phosphorylated 100

ABL1(F317L)-

nonphosphorylated 81

ABL1(F317L)-

phosphorylated 61

ABL1(H396P)-

nonphosphorylated 9

ABL1(H396P)-

phosphorylated 29

ABL1(M351T)-

phosphorylated 38

ABL1(Q252H)-

nonphosphorylated 20

ABL1(Q252H)-

phosphorylated 24

ABL1(T315I)-

nonphosphorylated 79

ABL1(T315I)-phosphorylated 73

ABL1(Y253F)-

phosphorylated 12

ABL1-nonphosphorylated 35

ABL1-phosphorylated 32

ABL2 56

ACVR1 15

ACVR1B 75

ACVR2A 70

ACVR2B 85

ACVRL1 52

ADCK3 81

ADCK4 24

AKT1 71

AKT2 62

AKT3 69

ALK 62

ALK(C1156Y) 37

ALK(L1196M) 57

AMPK-alpha1 15

AMPK-alpha2 23

ANKK1 47

ARK5 16

ASK1 100

ASK2 100

S12

AURKA 5.9

AURKB 23

AURKC 5.5

AXL 4

BIKE 21

BLK 5.8

BMPR1A 73

BMPR1B 47

BMPR2 40

BMX 82

BRAF 69

BRAF(V600E) 52

BRK 80

BRSK1 84

BRSK2 99

BTK 36

BUB1 59

CAMK1 53

CAMK1B 53

CAMK1D 72

CAMK1G 81

CAMK2A 84

CAMK2B 94

CAMK2D 92

CAMK2G 80

CAMK4 100

CAMKK1 68

CAMKK2 69

CASK 76

CDC2L1 80

CDC2L2 98

CDC2L5 100

CDK11 58

CDK2 81

CDK3 76

CDK4 100

CDK4-cyclinD1 100

CDK4-cyclinD3 86

CDK5 81

CDK7 46

CDK8 66

S13

CDK9 63

CDKL1 70

CDKL2 64

CDKL3 100

CDKL5 38

CHEK1 94

CHEK2 97

CIT 42

CLK1 1.1

CLK2 15

CLK3 57

CLK4 1

CSF1R 0.15

CSF1R-autoinhibited 0

CSK 88

CSNK1A1 71

CSNK1A1L 92

CSNK1D 55

CSNK1E 11

CSNK1G1 89

CSNK1G2 80

CSNK1G3 100

CSNK2A1 72

CSNK2A2 65

CTK 71

DAPK1 90

DAPK2 84

DAPK3 85

DCAMKL1 92

DCAMKL2 100

DCAMKL3 80

DDR1 27

DDR2 86

DLK 67

DMPK 100

DMPK2 100

DRAK1 47

DRAK2 77

DYRK1A 26

DYRK1B 38

DYRK2 78

S14

EGFR 56

EGFR(E746-A750del) 38

EGFR(G719C) 70

EGFR(G719S) 68

EGFR(L747-E749del,

A750P) 36

EGFR(L747-S752del, P753S) 42

EGFR(L747-T751del,Sins) 47

EGFR(L858R) 58

EGFR(L858R,T790M) 29

EGFR(L861Q) 53

EGFR(S752-I759del) 69

EGFR(T790M) 33

EIF2AK1 87

EPHA1 51

EPHA2 83

EPHA3 68

EPHA4 100

EPHA5 100

EPHA6 96

EPHA7 100

EPHA8 100

EPHB1 81

EPHB2 90

EPHB3 89

EPHB4 89

EPHB6 1.6

ERBB2 51

ERBB3 84

ERBB4 85

ERK1 100

ERK2 92

ERK3 100

ERK4 95

ERK5 92

ERK8 62

ERN1 44

FAK 67

FER 83

FES 80

FGFR1 14

S15

FGFR2 22

FGFR3 60

FGFR3(G697C) 51

FGFR4 94

FGR 14

FLT1 71

FLT3 0.35

FLT3(D835H) 1.6

FLT3(D835V) 0

FLT3(D835Y) 1.4

FLT3(ITD) 0.35

FLT3(ITD,D835V) 0

FLT3(ITD,F691L) 2.1

FLT3(K663Q) 1.1

FLT3(N841I) 1.6

FLT3(R834Q) 5.5

FLT3-autoinhibited 3.6

FLT4 18

FRK 53

FYN 45

GAK 42

GCN2(Kin.Dom.2,S808G) 100

GRK1 44

GRK2 100

GRK3 99

GRK4 56

GRK7 16

GSK3A 65

GSK3B 89

HASPIN 23

HCK 3.1

HIPK1 56

HIPK2 53

HIPK3 57

HIPK4 22

HPK1 39

HUNK 100

ICK 50

IGF1R 67

IKK-alpha 87

IKK-beta 89

S16

IKK-epsilon 41

INSR 27

INSRR 74

IRAK1 9.9

IRAK3 4.5

IRAK4 34

ITK 80

JAK1(JH1domain-catalytic) 14

JAK1(JH2domain-

pseudokinase) 0.2

JAK2(JH1domain-catalytic) 0.05

JAK3(JH1domain-catalytic) 6.4

JNK1 24

JNK2 74

JNK3 51

KIT 3.8

KIT(A829P) 27

KIT(D816H) 13

KIT(D816V) 0.75

KIT(L576P) 0

KIT(V559D) 1.2

KIT(V559D,T670I) 29

KIT(V559D,V654A) 43

KIT-autoinhibited 43

LATS1 19

LATS2 12

LCK 6.6

LIMK1 56

LIMK2 45

LKB1 100

LOK 38

LRRK2 49

LRRK2(G2019S) 61

LTK 68

LYN 48

LZK 83

MAK 61

MAP3K1 79

MAP3K15 77

MAP3K2 48

MAP3K3 41

S17

MAP3K4 88

MAP4K2 67

MAP4K3 83

MAP4K4 26

MAP4K5 91

MAPKAPK2 100

MAPKAPK5 100

MARK1 90

MARK2 81

MARK3 99

MARK4 80

MAST1 95

MEK1 79

MEK2 83

MEK3 32

MEK4 64

MEK5 23

MEK6 79

MELK 84

MERTK 4.6

MET 43

MET(M1250T) 29

MET(Y1235D) 73

MINK 4.8

MKK7 100

MKNK1 89

MKNK2 33

MLCK 100

MLK1 19

MLK2 49

MLK3 30

MRCKA 100

MRCKB 99

MST1 97

MST1R 78

MST2 12

MST3 93

MST4 83

MTOR 94

MUSK 86

MYLK 62

S18

MYLK2 70

MYLK4 51

MYO3A 99

MYO3B 100

NDR1 89

NDR2 100

NEK1 91

NEK10 1.6

NEK11 100

NEK2 97

NEK3 56

NEK4 100

NEK5 49

NEK6 71

NEK7 79

NEK9 95

NIK 96

NIM1 88

NLK 67

OSR1 100

p38-alpha 85

p38-beta 89

p38-delta 86

p38-gamma 84

PAK1 85

PAK2 56

PAK3 38

PAK4 83

PAK6 60

PAK7 76

PCTK1 95

PCTK2 72

PCTK3 100

PDGFRA 11

PDGFRB 0

PDPK1 100

PFCDPK1(P.falciparum) 24

PFPK5(P.falciparum) 100

PFTAIRE2 76

PFTK1 90

PHKG1 75

S19

PHKG2 71

PIK3C2B 81

PIK3C2G 51

PIK3CA 100

PIK3CA(C420R) 73

PIK3CA(E542K) 88

PIK3CA(E545A) 80

PIK3CA(E545K) 94

PIK3CA(H1047L) 100

PIK3CA(H1047Y) 89

PIK3CA(I800L) 77

PIK3CA(M1043I) 84

PIK3CA(Q546K) 100

PIK3CB 100

PIK3CD 88

PIK3CG 69

PIK4CB 1.1

PIKFYVE 29

PIM1 42

PIM2 45

PIM3 71

PIP5K1A 71

PIP5K1C 0

PIP5K2B 27

PIP5K2C 21

PKAC-alpha 50

PKAC-beta 56

PKMYT1 96

PKN1 64

PKN2 43

PKNB(M.tuberculosis) 89

PLK1 78

PLK2 100

PLK3 78

PLK4 34

PRKCD 45

PRKCE 46

PRKCH 73

PRKCI 78

PRKCQ 8.3

PRKD1 11

S20

PRKD2 19

PRKD3 7.4

PRKG1 100

PRKG2 74

PRKR 100

PRKX 80

PRP4 100

PYK2 63

QSK 83

RAF1 94

RET 14

RET(M918T) 2.1

RET(V804L) 10

RET(V804M) 8.8

RIOK1 26

RIOK2 37

RIOK3 80

RIPK1 98

RIPK2 39

RIPK4 53

RIPK5 75

ROCK1 10

ROCK2 25

ROS1 20

RPS6KA4(Kin.Dom.1-N-

terminal) 78

RPS6KA4(Kin.Dom.2-C-

terminal) 100

RPS6KA5(Kin.Dom.1-N-

terminal) 100

RPS6KA5(Kin.Dom.2-C-

terminal) 100

RSK1(Kin.Dom.1-N-

terminal) 9.9

RSK1(Kin.Dom.2-C-

terminal) 99

RSK2(Kin.Dom.1-N-

terminal) 42

RSK2(Kin.Dom.2-C-

terminal) 79

RSK3(Kin.Dom.1-N-

terminal) 44

RSK3(Kin.Dom.2-C- 100

S21

terminal)

RSK4(Kin.Dom.1-N-

terminal) 50

RSK4(Kin.Dom.2-C-

terminal) 87

S6K1 64

SBK1 66

SGK 92

SgK110 33

SGK2 95

SGK3 58

SIK 46

SIK2 73

SLK 19

SNARK 17

SNRK 69

SRC 2.2

SRMS 71

SRPK1 30

SRPK2 100

SRPK3 55

STK16 42

STK33 44

STK35 86

STK36 49

STK39 97

SYK 16

TAK1 19

TAOK1 97

TAOK2 94

TAOK3 98

TBK1 62

TEC 94

TESK1 77

TGFBR1 75

TGFBR2 30

TIE1 17

TIE2 38

TLK1 100

TLK2 100

TNIK 33

TNK1 40

S22

TNK2 43

TNNI3K 95

TRKA 5.3

TRKB 16

TRKC 27

TRPM6 97

TSSK1B 60

TSSK3 86

TTK 30

TXK 20

TYK2(JH1domain-catalytic) 0.2

TYK2(JH2domain-

pseudokinase) 3.8

TYRO3 9.3

ULK1 100

ULK2 80

ULK3 44

VEGFR2 69

VPS34 21

VRK2 82

WEE1 90

WEE2 100

WNK1 87

WNK2 100

WNK3 94

WNK4 100

YANK1 69

YANK2 100

YANK3 55

YES 28

YSK1 100

YSK4 18

ZAK 93

ZAP70 91 aPercent of Control: normalized % control based on the negative control (DMSO,100% control)

and positive control (control compound, 0% control). %Ctrl is calculated using the following

equation:

[(test compound signal-positive ctrl signal)/(negative ctrl signal-positive ctrl signal)]x100

S23

Table S2. FLT3 and MV4-11 data with confidence intervalsa

Compd

FLT3

IC50

(nM)

FLT3

CI 95

(nM)

MV4-

11

EC50

(nM)

MV4-11

CI 95

(nM)

MOLM13F

LT3-ITD EC50

(nM)

MOLM13F

LT3-ITD

CI 95 (nM)

MOLM

13 FLT3-

ITD/D835Y

EC50

(nM)

MOLM

13 FLT3-

ITD/D835Y

CI 95

(nM)

1 32 24-43 320 160-650 620 461-833 1153

869-

1604

4a 1890 670-5360

>700

0 - N.D.b N.D. N.D. N.D.

4b

>130

00 -

>500

0 - N.D. N.D. N.D. N.D.

4c 2590 570-11690

>150

00 - N.D. N.D. N.D. N.D.

4d 379 177-811 5050

3770-

6750 N.D. N.D. N.D. N.D.

4e

>190

00 -

>700

0 - N.D. N.D. N.D. N.D.

4f 978 354-2703 5230

2380-

11490 N.D. N.D. N.D. N.D.

4g

>120

00 -

>400

0 - N.D. N.D. N.D. N.D.

4h 1710 1130-2950 3230

2390-

4370 N.D. N.D. N.D. N.D.

4i 24500

5700-

104700

>150

00 - N.D. N.D. N.D. N.D.

4j 13300

7600-

56200 7590

1390-

41320 N.D. N.D. N.D. N.D.

4k 336 179-630 511 18-14512 N.D. N.D. N.D. N.D.

4l 96 73-125

>140

00 - N.D. N.D. N.D. N.D.

4m 16 11-21 58 32-181 N.D. N.D. N.D. N.D.

4n 20 14-28 53 11-253 N.D. N.D. N.D. N.D.

4o 722 260-2010 369 146-940 N.D. N.D. N.D. N.D.

4p 3260 740-14290 3570

660-

19350 N.D. N.D. N.D. N.D.

4q

>230

00 -

>800

0 - N.D. N.D. N.D. N.D.

4r 3300 940-11620

>300

0 - N.D. N.D. N.D. N.D.

4s 14 9.2-20.9 113 15-872 N.D. N.D. N.D. N.D.

4t 1880 880-4030 41 0.2-6742 N.D. N.D. N.D. N.D.

4u

>310

00 - 1260 330-4790 N.D. N.D. N.D. N.D.

5a 204 72-570

>200

00 - N.D. N.D. N.D. N.D.

5b 47.5 32.6-69.2 35 16-78 N.D. N.D. N.D. N.D.

S24

5c <6 - 138 66-287 N.D. N.D. N.D. N.D.

5d 123 62-246 340 174-667 N.D. N.D. N.D. N.D.

5e <6 - 25 1-603 136 112-164 82 66-103

5f 13 11-16 25 13-49 N.D. N.D. N.D. N.D.

5g <6 -

>230

00 - N.D. N.D. N.D. N.D.

5h

>220

00 -

>800

0 - N.D. N.D. N.D. N.D.

5i

>230

00 -

>800

0 - N.D. N.D. N.D. N.D.

5j 8 6-11 65 9-487 N.D. N.D. N.D. N.D.

5k <6 - 25 17-39 N.D. N.D. N.D. N.D.

5l <6 - 141 89-287 N.D. N.D. N.D. N.D.

5m <6 - 31 6-168 N.D. N.D. N.D. N.D.

5n <6 -

>170

00 - N.D. N.D. N.D. N.D.

5o 13 7-24 58 44-86 N.D. N.D. N.D. N.D.

5p

>340

00 -

>120

00 - N.D. N.D. N.D. N.D.

5q 8 4-19 654 219-1951 N.D. N.D. N.D. N.D.

5r 185 99-343 1877 158-4643 N.D. N.D. N.D. N.D.

6a

>240

00 -

>160

00 - >10000 - 4260

3060-

5930

6b 17200

8900-

33300

>130

00 - N.D. N.D. N.D. N.D.

6c 11600

6100-

22300 3980

3120-

5070 N.D. N.D. N.D. N.D.

6d 2880 850-9740 4820

1390-

16740 N.D. N.D. N.D. N.D.

6e 6970

3940-

12310

>160

00 - N.D. N.D. N.D. N.D.

6f 4440

1060-

18570 4890

270-

92980 N.D. N.D. N.D. N.D.

6g 918 301-2801 1060 680-1660 N.D. N.D. N.D. N.D.

6h 475 270-837 9460

4850-

18470 N.D. N.D. N.D. N.D.

6i 42 33-54 459 49-4235 N.D. N.D. N.D. N.D.

6j <6 - 4 2-7 N.D. N.D. N.D. N.D.

6k 11 5-28 5 3-9 45 38-55 77 63-94

6l 284 75-1074

>150

00 - N.D. N.D. N.D. N.D.

6m 15000

8300-

27300 11900

3500-

40700 N.D. N.D. N.D. N.D.

6n 213 118-384 2530 690-9230 N.D. N.D. N.D. N.D.

9 80 21-314

>100

00 - N.D. N.D. N.D. N.D.

S25

13 1500 530-4220

>160

00 - N.D. N.D. N.D. N.D.

14 141 12-1649

>800

0 - N.D. N.D. N.D. N.D.

16 82 42.3-159

>800

0 - N.D. N.D. N.D. N.D.

17

>150

00 - 39 13-118 N.D. N.D. N.D. N.D.

19 187 82-600

>600

0 - N.D. N.D. N.D. N.D.

20 49 27-87

>130

00 - N.D. N.D. N.D. N.D.

21

>110

00 NA

>400

0 - N.D. N.D. N.D. N.D.

22 1260 340-4710

>140

00 - N.D. N.D. N.D. N.D.

Quizart-

inib 40 15-111 0.7 0.2-3.1 N.D. N.D. N.D. N.D. aValues are reported as the mean of triplicate experiments with 95% confidence intervals (CI 95)

bN.D. means not determined

Table S3. BJ growth inhibition data of FLT3 inhibitorsa

Compd

BJ EC50

(µM) CI 95 (µM)

1 >9 -

4a >11 -

4b >7 -

4c 12.2294 6.1188-24.4422

4d >20 -

4e >10 -

4f >17 -

4g >5 -

4h 12.0608 9.6321-15.1018

4i 12.4731 8.0953-19.2184

4j 21.498 16.4970-28.0150

4k >45 -

4l >15 -

4m >8 -

4n >12 -

4o >14 -

4p >11 -

4q >8 -

4r >6 -

S26

4s >13 -

4t 9.1788 7.9173-10.6414

4u >13 -

5a >20 -

5b >21 -

5c >19 -

5d >17 -

5e >17 -

5f >20 -

5g >23 -

5h >8 -

5i >9 -

5j >6 -

5k >6 -

5l >19 -

5m >7 -

5n >17 -

5o 11 10.6123-13.0892

5p >12 -

5q >10 -

5r >24 -

6a >10 -

6b >14 -

6c >20 -

6d >13 -

6e 13.9205 7.5059-25.8173

6f 13.2277 10.9040-16.0467

6g >19 -

6h >21 -

6i >36 -

6j >16 -

6k >18 -

6l >16 -

6m >26 -

6n >12 -

9 >10 -

13 10.615 9.3450-12.0575

14 8.7406 6.9389-11.0103

16 >8 -

17 >5 -

19 >6 -

S27

20 >13 -

21 >4 -

22 >14 - aValues are the mean of a single triplicate experiment

Table S4. Solubility and PAMPA permeability of selected compounds

Compound

Solubility

(µM)

Permeability (10-6

cm/s)a

pH = 7.4a

1 0.4 ± 0.9 1880 ± 170

4g < 0.1 undetected

4h 1.0 ± 4.4 1530 ± 150

4j 0.4 ± 1.8 76.9

4i 0.3 ± 1.1 undetected

5a 57.9 ± 2.2 2250 ± 70

5b 50.70 ± 0.7 2200 ± 100

5c 29.4 ± 1.5 equilibrated

5d 5.0 ± 0.2 1720 ± 340

5e 26.4 ± 17.0 2000 ± 190

5f 13.1 ± 3.3 1990.2

5g 59.1 ± 3.6 1910 ± 190

5h 52.6 ± 1.5 1910 ± 150

5i 61.9 ± 2.2 9.7 ± 3.3

5j 15.9 ± 3.0 2000 ± 170

5k 17.5 ± 1.9 equilibrated

5l 39.8 ± 3.5 equilibrated

5n 7.6 ± 0.4 2050 ± 280

5o 2.0 ± 0.2 equilibrated

5p 57.7 ± 0.8 1300 ± 50

5q 68.4 ± 2.0 8.2 ± 4.8

5r 64.1 ± 3.1 1840 ± 30

6a 26.5 ± 1.1 2360 ± 150

6b 10.1 ± 0.4 1602.4

6c 0.38 ± 0.1 equilibrated

6d 24 ± 29.3 2010 ± 130

6e 0.2 ± 0.7 equilibrated

6g 53.8 ± 3.2 equilibrated

6h 29.6 ± 5.1 1960 ± 80

6i 66.7 ± 2.0 1740 ± 210

6j 51.9 ± 2.9 2069 ± 3

S28

6k 56.9 ± 1.9 1870 ± 60

13 58.9 ± 0.5 equilibrated aValues are the mean of a single triplicate experiment.

Table S5. PGP Efflux susceptiblity of selected analogs

MDR1-MDCKII Permeabilitya MDCKII-WT Permeability

a

Compd

AVG Papp A/B

(nm/s)b

AVG Papp B/A

(nm/s)b

Efflux

Ratioc

(B2A/A2

B)

AVG Papp A/B

(nm/s)b

AVG Papp B/A

(nm/s)b

Efflux

Ratioc

(B2A/A2

B)

1 103.68 ± 49.25 105.59 ± 40.39 1.02 70.04 ± 31.18 75.49 ± 27.71 1.08

4g 0.04 ± 0.01 0.28 ± 0.21 7.33 0.05 ± 0.04 0.69 ± 0.08 14.77

4h 157.21 ± 89.03

185.77 ±

146.66 1.18 173.38 ± 65.84

151.41 ±

120.59 0.87

4i 0.16 ± 0.18 0.94 ± 0.42 5.96 0.19 ± 0.16 1.97 ± 1.34 10.35

4j 0.15 ± 0.11 0.76 ± 0.54 5.23 0.17 ± 0.05 1.44 ± 0.75 8.30

5e

801.70 ±

421.18

616.01 ±

260.74 0.77 750.19 ± 390.86

507.21 ±

202.15 0.68

6a 184.18 ± 12.05 150.48 ± 51.89 0.82 172.23 ± 21.61 120.77 ± 29.64 0.70

6b 19.40 ± 6.60 15.70 ± 6.80 0.81 15.90 ± 6.60 20.90 ± 12.20 1.31

6c 6.19 ± 0.64 8.86 ± 2.17 1.43 6.38 ± 1.25 14.83 ± 2.81 2.33

6d 3.90 ± 2.22 10.35 ± 7.07 2.66 3.88 ± 2.90 11.61 ± 8.78 2.99

6e 3.69 ± 0.49 7.90 ± 2.35 2.14 3.27 ± 1.19 5.43 ± 0.56 1.66

6l

751.63 ±

361.85

537.83 ±

252.38 0.72 685.83 ± 321.76

407.00 ±

200.55 0.59 aMDR1-MDCKII cell line over-expresses PGP transporter.

bValues are the mean of a single triplicate experiment.

cEfflux ratio >2 indicates compound may be susceptible to PGP efflux.

Table S6. In vivo blood chemistry data after i.p. dosing of 5e.

Dose (mg/kg) 0 3 5 10

Albumin (g/dl) 3.9 3.3 3.9 3.6

Alk Phos (U/L) 159 144 207 147

ALT (U/L) 196.2 101.1 45 46.5

Amylase (U/L) 3426 2331 3663 3765

BUN (mg/dl) 24.9 16.8 26.7 24.3

Calcium (mg/dl) 9.81 10.05 10.11 8.7

Creatinine (mg/dl) 0.3 0.3 0.24 0.3

Glucose (mg/dl) 132.9 210.3 228.6 132.6

Potassium (mmol/L) 8.28 8.52 9.69 7.32

Sodium (mmol/L) 159 156 159 159

S29

Phosphorus (mg/dl) 11.7 10.5 12 11.4

Total Bilirubin

(mg/dl) 0.3 0.27 0.3 0.21

Total Protein (g/dl) 6.3 5.1 5.7 5.4

Globulin (g/dl) 2.4 1.8 1.8 1.8

References

1. Uchida, M.; Fukazawa, T.; Yamazaki, Y.; Hashimoto, H.; Miyamoto, Y. A modified fast

(4 day) 96-well plate Caco-2 permeability assay. J Pharmacol Toxicol Methods 2009, 59, 39-43.

2. Larson, B.; Banks, P.; Sherman, H.; Rothenberg, M. Automation of cell-based drug

absorption assays in 96-well format using permeable support systems. J Lab Autom 2012, 17,

222-232.

3. Albrecht, F.; Sowada, O.; Fistikci, M.; Boysen, M. M. K. Heteroarylboronates in

Rhodium-Catalyzed 1,4-Addition to Enones. Organic Letters 2014, 16, 5212-5215.

4. Bhattacharyya, S.; Neidigh, K. A.; Avery, M. A.; Williamson, J. S. Selective

monoalkylation of ammonia: A high throughput synthesis of primary amines. Synlett 1999,

1781-1783.