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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.
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