Potent and selective mitogen-activated protein kinase kinase (MEK) 1,2 inhibitors. 1. 4-(4-bromo-2-fluorophenylamino)-1-methylpyridin-2(1h)-ones

Article abstract of DOI:10.1021/jm050834y

The role of MEK 1,2 in cancer tumorgenesis has been clearly demonstrated preclinically, and two selective inhibitors are currently undergoing clinical evaluation to determine their role in the human disease. We have discovered 4-(4-bromo-2-fluorophenylamino)-1-methylpyridin-2(1H)-ones as a new class of ATP noncompetitive MEK inhibitors. These inhibitors exhibit excellent cellular potency and good pharmacokinetic properties and have demonstrated the ability to inhibit ERK phosphorylation in HT-29 tumors from mouse xenograft studies.

Full text of DOI:10.1021/jm050834y

Supporting Information
Potent and Selective MEK 1,2 Inhibitors. Part 1: 4-(4-Bromo-2-fluorophenylamino)-
1-methyl-pyridin-2(1H)-ones
Eli M. Wallace*, Joseph Lyssikatos, James F. Blake, Jeongbeob Seo, Hong Woon Yang,
Tammie C. Yeh, Michele Perrier, Heidi Jarski, Vivienne Marsh, Gregory Poch, Michelle
Goyette Livingston, Jennifer Otten, Gary Hingorani, Rich Woessner, Patrice Lee, James
Winkler, and Kevin Koch
Array BioPharma, 3200 Walnut Street, Boulder, CO, USA
Table of Contents
Contents
Chemistry Experimental Section
Page Number
S1 – S13
Elemental Analysis Data for Compounds 13, 16, 22, 23, and 24 S14
Biological Assay Details
Selectivity Data
S15
S16
Non-Competitive Inhibition of 16
Hepatocyte Assay Details
Caco-2 Assay Details
Solubility Assay Details
Rodent PK Details
S17
S18
S19-20
S21
S22
PK/PD Description
S23-25
Chemistry Experimental Section
The reactions set forth below were done generally under a positive pressure of nitrogen or
argon or with a drying tube (unless otherwise stated) in anhydrous solvents, and the
reaction flasks were typically fitted with rubber septa for the introduction of substrates
and reagents via syringe or cannula. Glassware was oven dried and/or heat dried. All
reagents and solvents were used without further purification unless otherwise stated.
Reactions were monitored by either analytical TLC or analytical HPLC. Analytical TLC
was performed using glass plates pre-coated with silica gel (Manufacturer: EMD, Silica
Gel 60 F254, 250 µm). Analytical HPLC was performed on YMC ODS-AQ 3 µm, 120 ,
3.0 x 50 mm column using a 0.01% HFBA/1% IPA/water/acetonitrile gradient and UV
detection at 220 and 254 nm. Flash column chromatography was performed on a Biotage
system (Manufacturer: Dyax Corporation) having pre-packed silica gel columns
S1

(Manufacturer: Biotage, part no. FPK0-1107-15046, FPK0-1107-17026, FPK0-1107-
17046, or FK0-1107-1804C or on Isolute SPE columns (Manufacturer: International
Sorbent Technology, part no. 450-0500-E or 450-1000-F). Mass spectra were recorded
on Thermo Finnigan LCQ Duo Flow Injection APCI ( ). Combustion analyses were
performed by Schwarzkopf Microanalytical Laboratory, Inc. (Woodside, NY). All
analyzed compounds are within 0.4% of the theoretical value unless otherwise
indicated. 1H-NMR spectra were recorded on a Varian Mercury (400 MHz) NMR
spectrometer. 19F-NMR spectra were recorded on a Varian Mercury (376 MHz) NMR
spectrometer. Chemical shifts are expressed in parts per million (ppm, scale) using
tetramethylsilane as the reference standard. When peak multiplicities are reported, the
following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), dd (doublet of doublet), dt (doublet of triplet), br (broad). Coupling constants
are reported in Hertz (Hz).
4-(4-bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (6).
Step A: Preparation of ethyl 4-hydroxy-1-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylate: To diethyl 1,3-acetonedicarboxylate (50.0 mL, 276 mmol) was added
triethyl orthoformate (45.8 mL, 276 mmol) followed by acetic anhydride (52.1 mL, 551
o
mmol). The resulting mixture was stirred for 1 h at 135 C. The reaction mixture was
cooled to room temperature and concentrated under reduced pressure. To the resulting
o
residue at 0 C was added methylamine (53 mL, 690 mmol, 40% in water) followed by
water (200 mL) with stirring. The reaction mixture was stirred at room temperature for
16 h. The reaction mixture was diluted with EtOAc (300 mL). The organic layer was
separated. The aqueous phase was neutralized with 10% HCl solution to produce the
desired product as a white precipitate that was filtered and washed with water. The
filtrate was extracted with EtOAc. The combined organic extracts dried over MgSO4 and
concentrated under reduced pressure to give a white solid that was rinsed with Et2O and
combined with the first crop to yield 29 g (54%) of the desired product. MS APCI (+)
1
m/z 198 (M+) detected; H NMR (400 MHz, CD3OD) 8.48 (s, 1H), 5.82 (s, 1H), 4.40
(q, J = 7.2 Hz, 2H), 3.59 (s, 3H), 1.38 (t, J = 7.6 Hz, 3H).
S2

Step B: Preparation of ethyl 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate:
To a suspension of ethyl 4-hydroxy-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate
(10.0 g, 50.7 mmol) and POCl3 (27.8 mL, 304 mmol) was added triethylamine (7.07 mL,
50.7 mmol). After stirring for 16 h, the reaction mixture was concentrated under reduced
pressure to remove POCl3. The resulting residue was poured onto ice, carefully
neutralized with sat’d aq K2CO3 solution, and extracted with EtOAc. The combined
organic extracts were dried over MgSO4 and concentrated under reduced pressure to yield
7.3 g (67%) of the desired product that was used directly without further purification.
1
MS APCI (+) m/z 216, 218 (M+, Cl pattern) detected; H NMR (400 MHz, CD3OD)
8.49 (s, 1H), 6.62 (s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 3.59 (s, 3H), 1.36 (t, J = 7.6 Hz, 3H).
Step C: Preparation of 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid:
To a solution of ethyl 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (925
mg, 4.29 mmol) in a 4:1 mixture of THF-MeOH (20 mL) was added 1 N aq LiOH (8.6
mL, 8.6 mmol) at room temperature. After stirring for 30 minutes, the reaction mixture
was acidified to pH 1 with 10% aq HCl and extracted with EtOAc. The combined
organic extracts were washed with brine, dried over MgSO4, and concentrated under
reduced pressure to give 0.732 g (91%) of the desired product that was used directly
1
without further purification. MS APCI (-) m/z 186, 188 (M-, Cl pattern) detected; H
NMR (400 MHz, CD3OD) 8.53 (s, 1H), 6.62 (s, 1H), 3.58 (s, 3H).
Step D: Preparation of 4-(4-bromo-2-fluorophenylamino)-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxylic acid: To a solution of i-Pr2NH (0.39 mL, 2.80 mmol) in
THF (4 mL) at 0 oC was added n-BuLi (1.10 mL, 2.80 mmol, 2.5 M solution in hexanes).
o
After stirring for 15 min, the resulting mixture was cooled to -78 C. 4-Bromo-2-
fluoroaniline (0.38 g, 2.0 mmol) was added. After vigorous stirring for 10 minutes, a
mixture of the 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid (0.15 g,
0.80 mmol) in THF (5 mL) was added. After stirring for 30 min at –78 oC, the dry-ice
bath was removed. The reaction mixture was slowly warmed to room temperature and
stirred for 17 h. The reaction mixture was treated with 10% aq HCl (15 mL), extracted
with EtOAc, dried over MgSO4, and concentrated under reduced pressure to give the
crude material that was triturated with CH2Cl2 to afford 0.21 g (77%) of the desired
1
product. MS APCI (-) m/z 339, 341 (M-, Br pattern) detected; H NMR (400 MHz,
S3

DMSO-d6) 9.61 (s, 1H), 8.53 (s, 1H), 7.69 (dd, J = 10.8, 2.0 Hz, 1H), 7.46 (m, 2H),
5.47 (s, 1H), 3.41 (s, 3H); 19F NMR (376 MHz, DMSO-d6) –121.4.
Step E: Preparation of 4-(4-bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-1-
methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (6): A mixture of 4-(4-bromo-2-
fluorophenylamino)-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid (78 mg, 0.23
mmol), EDCI (130 mg, 0.69 mmol), and HOBt (93 mg, 0.69 mmol) in DMF (5 ml) was
stirred for 30 minutes at room temperature. O-Cyclopropylmethylhydroxylamine (60 mg,
0.69 mmol) was added followed by Et3N (0.096 mL, 0.69 mmol). After stirring for 1 h,
the reaction mixture was diluted with EtOAc and washed with sat’d aq NH4Cl, sat’d aq
NaHCO3, and brine. The organic layer was dried over MgSO4 and concentrated under
reduced pressure to yield 83 mg (89%) of the desired product. MS APCI (+) m/z 410,
1
412 (M+, Br pattern) detected; H NMR (400 MHz, DMSO-d6) 11.57 (s, 1H), 9.42 (s,
1H), 8.10 (s, 1H), 7.67 (d, J = 10.4 Hz, 1H), 7.43 (m, 2H), 3.70 (d, J = 7.2 Hz, 2H), 3.35
(s, 3H), 1.11 (m, 1H), 0.54 (m, 2H), 0.27 (m, 2H); 19F NMR (376 MHz, DMSO-d6)
–
121.8.
4-(4-Bromo-2-fluorophenylamino)-5-chloro-N-(cyclopropylmethoxy)-1-methyl-6-
oxo-1,6-dihydropyridine-3-carboxamide (7).
Preparation of 7 is as described for 6 with the addition of the following chlorination step
after Step B.
Preparation of ethyl 4,5-dichloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate: A
mixture of ethyl 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (1.00 g,
4.64 mmol) and NCS (0.68 g, 5.10 mmol) in DMF (30 mL) was stirred for 1 h at room
temperature. The reaction mixture was diluted with EtOAc and washed with 0.1 N aq
HCl. The organic layer was dried over MgSO4 and concentrated under reduced pressure
to give 1.10 g (95%) of the desired product that was used directly without further
purification. MS APCI (+) m/z 250, 252 (M+, Cl pattern) detected; 1H NMR (400 MHz,
CDCl3) 8.14 (s, 1H), 4.36 (q, J = 7.2 Hz, 2H), 3.67 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H).
4-(4-Bromo-2-fluorophenylamino)-5-chloro-N-(cyclopropylmethoxy)-1-methyl-6-oxo-
1,6-dihydropyridine-3-carboxamide (7): MS APCI (+) m/z 444, 446 (M+, Cl, Br pattern)
detected; 1H NMR (400 MHz, DMSO-d6) 11.52 (s, 1H), 8.78 (s, 1H), 7.98 (s, 1H), 7.52
(dd, J = 10.4, 2.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.93 (dd, J = 8.4, 8.4 Hz, 1H), 3.47
S4

19
(s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 1.01 (m, 1H), 0.50 (m, 2H), 0.20 (m, 2H); F NMR
(376 MHz, DMSO-d6) –122.4.
4-(4-Bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-5-fluoro-1-methyl-6-
oxo-1,6-dihydropyridine-3-carboxamide (8).
Preparation of 8 is as described for 6 with the addition of the following fluorination step
after Step B.
Preparation of 4-chloro-5-fluoro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid:
A mixture of ethyl 4-chloro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (1.00 g,
4.64 mmol) and Selectfluor [1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane-
bis(tetrafluoroborate)] (3.29 g, 9.27 mmol) in MeCN (50 mL) was stirred at 85 oC for 1 h.
The reaction was cooled to room temperature and concentrated under reduced pressure.
The residue was dissolved in water and extracted with EtOAc. The combined organic
extracts were dried over MgSO4 and concentrated under reduced pressure to give the
crude material that was purified by silica gel flash column chromatography (40% EtOAc
in hexanes) to give the partially purified ethyl ester. To a solution of the partially
purified ethyl ester in a 4:1 mixture of THF:MeOH (10 mL) was added 1 N aq LiOH
(9.30 mL, 9.30 mmol) at room temperature. After stirring for 1 h, the reaction mixture
was acidified to pH 1 with 10% aq HCl and extracted with EtOAc. The combined
organic extracts were washed with brine, dried over MgSO4, and concentrated under
reduced pressure to give the crude material that was triturated with diethyl ether to yield
0.25 g (26% for two steps) of 4-chloro-5-fluoro-1-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylic acid. 1H NMR (400 MHz, DMSO-d6) 8.48 (s, 1H), 3.56 (s, 3H); 19F NMR
(376 MHz, DMSO-d6) –133.9.
4-(4-Bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-5-fluoro-1-methyl-6-oxo-
1,6-dihydropyridine-3-carboxamide (8): MS APCI (+) m/z 428, 430 (M+, Br pattern)
1
detected; H NMR (400 MHz, CD3OD) 7.85 (s, 1H), 7.36 (dd, J = 10.8, 2.0 Hz, 1H),
7.28 (d, J = 8.4 Hz, 1H), 7.01 (dt, J = 8.8, 4.4 Hz, 1H), 3.69 (d, 2H), 3.57 (s, 3H), 0.89
(m, 1H), 0.58 (m, 2H), 0.30 (m, 2H); 19F NMR (376 MHz, CD3OD) –125.8, –152.1.
4-(4-Bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (9).
Preparation of ethyl 4-hydroxy-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxylate:
S5

To diethyl 2-methyl-3-oxopentanedioate (Caliskan et al Aust. J. Chem. 1999, 52, 1013-
1020) (5.00 g, 23.1 mmol) was added triethyl orthoformate (3.85 mL, 23.1 mmol) and
acetic anhydride (4.37 mL, 46.2 mmol) at room temperature. The resulting mixture was
o
stirred for 1 h at 135 C. The reaction mixture was cooled to room temperature and
o
concentrated under reduced pressure. To the resulting residue 0 C was added
methylamine (5.00 mL, 57.8 mmol, 40% in water) followed by water (20 mL). The
reaction mixture was warmed to room temperature and stirred for 16 h. The reaction
mixture was extracted with EtOAc. The aqueous layer was acidified to pH 2 with 10%
aq HCl. The acidified aqueous layer was extracted with EtOAc. The combined organic
extracts were dried over MgSO4 and concentrated under reduced pressure to give the
crude material that was purified by trituration with diethyl ether to yield 4.88 g (55%) of
the desired product. MS APCI (+) m/z 212 (M+) detected; 1H NMR (400 MHz, CD3OD)
8.39 (s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.57 (s, 3H), 1.97 (s, 3H), 1.39 (t, J = 7.2 Hz,
3H).
Compound 9 was prepared as described in Steps B – E for compound 6.
4-(4-Bromo-2-fluorophenylamino)-N-(cyclopropylmethoxy)-1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (9): MS APCI (+) m/z 424, 426 (M+, Br pattern)
1
detected; H NMR (400 MHz, CD3OD) 7.88 (s, 1H), 7.33 (dd, J = 10.4, 1.2 Hz, 1H),
7.20 (d, J = 8.8 Hz, 1H), 6.65 (dd, J = 8.4, 8.4 Hz, 1H), 3.59 (d, J = 6.8 Hz, 2H), 3.57 (s,
3H), 1.83 (s, 3H), 1.10 (m, 1H), 0.54 (m, 2H), 0.25 (m, 2H); 19F NMR (376 MHz,
CD3OD) –128.2.
4-(4-Bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (10).
Step A:
Preparation of 4-(4-bromo-2-fluorophenylamino)-1-methyl-6-oxo-N-(2-
(vinyloxy)ethoxy)-1,6-dihydropyridine-3-carboxamide: A mixture of 4-(4-bromo-2-
fluorophenylamino)-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid (120 mg,
0.35 mmol), EDCI (100 mg, 0.53 mmol), and HOBt (71 mg, 0.53 mmol) in DMF (5 mL)
was stirred at room temperature for 3 h. O-(2-Vinyloxyethyl)hydroxylamine (0.071 mL,
0.70 mmol) was added followed by Et3N (0.098 mL, 0.70 mmol). After stirring for 2 h,
the reaction mixture was diluted with EtOAc and washed with sat’d aq NH4Cl, sat’d aq
S6

NaHCO3, and brine. The organic layer was dried over MgSO4 and concentrated under
reduced pressure to give the crude material that was purified by silica gel flash column
chromatography (3% MeOH in CH2Cl2) to afford 78 mg (52%) of the desired product.
The desired product was used without further characterization except MS. MS APCI (+)
m/z 426, 428 (M+, Br pattern) detected.
Step B:
Preparation of 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1-
methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (10): To a solution of 4-(4-bromo-2-
fluorophenylamino)-1-methyl-6-oxo-N-(2-(vinyloxy)ethoxy)-1,6-dihydropyridine-3-
carboxamide (77 mg, 0.18 mmol) in a 1:1 mixture of EtOH:THF (6 mL) was added 1 N
aq HCl (0.36 mL, 0.36 mmol) at room temperature. After stirring for 1 h, the pH of the
reaction mixture was adjusted to 5 to 7 with 2 N aq NaOH. The resulting mixture was
diluted with EtOAc, washed with water, dried over MgSO4, and concentrated under
reduced pressure to give the crude material that purified by trituration with diethyl ether
to yield 55 mg (76%) of the desired product. MS APCI (-) m/z 398, 400 (M-, Br pattern)
detected; 1H NMR (400 MHz, DMSO-d6) 11.65 (s, 1H), 9.41 (s, 1H), 8.13 (s, 1H), 7.68
(d, J = 9.6 Hz, 1H), 7.43 (m, 2H), 5.53 (s, 1H), 4.74 (m, 1H), 3.91 (t, J = 4.8 Hz, 2H),
3.62 (m, 2H), 3.36 (s, 3H); 19F NMR (376 MHz, DMSO-d6) –121.8.
4-(4-Bromo-2-chlorophenylamino)-5-chloro-N-(2-hydroxyethoxy)-6-
methoxynicotinamide (14).
Step A: Preparation of ethyl 5-chloro-4,6-dihydroxynicotinate: To a solution of ethyl
4,6-dihydroxynicotinate (20.0 g, 109 mmol, J. Heterocyclic Chem. 1983, 20, 1363) in
DMF (400 mL) at room temperature was portionwise added NCS (17.5 g, 131 mmol)
over 10 min. The resulting mixture was stirred for 3 days. After addition of sat’d aq
NaHCO3, the resulting mixture was stirred for 2 h at room temperature. The mixture was
concentrated under reduced pressure to give the crude material that was partitioned
between EtOAc and water. The precipitates were filtered off and dried under reduced
pressure to afford 6.12 g (26%) of the desired product. MS APCI (-) m/z 216, 218 (M-;
1
Cl pattern) detected. H NMR (400 MHz, DMSO-d6) 8.06 (s, 1H), 4.32 (q, J = 7.2 Hz,
2H), 1.30 (t, J = 7.2 Hz, 3H).
Step B: Preparation of ethyl 4,5,6-trichloronicotinate: To a suspension of ethyl 5-chloro-
4,6-dihydroxynicotinate (6.12 g, 28.1 mmol) in POCl3 (40 mL) was slowly added TEA
S7

(4.00 mL, 28.7 mmol) at room temperature. The resulting mixture was stirred for 4 h at
o
60 C. The reaction mixture was cooled to room temperature, poured onto ice, and
neutralized by sat’d aq K2CO3 and NaOH pellets. The resulting mixture was extracted
with EtOAc followed by Et2O, dried over Na2SO4, filtered, and concentrated under
reduced pressure to give the crude material that was purified by silica gel flash column
chromatography (EtOAc:hexanes = 1:5) to afford 6.04 g (84%) of the desired product.
1H NMR (400 MHz, CDCl3) 8.68 (s, 1H), 4.45 (q, J = 6.8 Hz, 2H), 1.42 (t, J = 6.8 Hz,
3H).
Step C: Preparation of 4,5,6-trichloronicotinic acid: To a solution of ethyl 4,5,6-
trichloronicotinate (7.76 g, 30.5 mmol) in a 4:1 mixture of THF-MeOH (150 mL) was
added 1 N aq NaOH (61 mL, 61 mmol) at room temperature. The reaction mixture was
stirred for 30 min, acidified to pH 1 with conc. HCl, and diluted with EtOAc. The
mixture was washed with water and brine. The organic layer was dried over Na2SO4,
filtered, and concentrated under reduced pressure to yield 6.77 g (98%) of the crude
1
product that was used directly without further purification. H NMR (400 MHz, CDCl3)
8.86 (s, 1H).
Step D: Preparation of 4-(4-bromo-2-chlorophenylamino)-5,6-dichloronicotinic acid: To
a solution of 4-bromo-2-chloroaniline (11.4 g, 55.3 mmol) in a 1:3 mixture of THF-
o
hexanes (92 mL) at –78 C was added LiHMDS (70.7 mL, 70.7 mmol, 1 M solution in
o
THF) over 7 min. The resulting mixture was stirred for 1 h at –78 C. A solution of
o
4,5,6-trichloronicotinic acid (5.12 g, 22.6 mmol) in THF (15 mL) was added at –78 C.
The reaction mixture was slowly warmed to room temperature and stirred for 3 h. The
reaction mixture was quenched with water and acidified to pH 1-2 with 2 N aq HCl. The
o
resulting mixture was cooled to 10 C with ice-water bath and stirred for 25 min. The
precipitates were filtered and dried under reduced pressure to afford 8.64 g (99%) of the
1
desired product. MS APCI (-) m/z 393, 395, 397 (M-; Cl, Br pattern) detected. H NMR
(400 MHz, DMSO-d6) 9.90 (s, 1H), 8.68 (s, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.48 (dd, J =
2.0, 8.8 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H).
Step E: Preparation of methyl 4-(4-bromo-2-chlorophenylamino)-5,6-dichloronicotinate:
To a suspension of 4-(4-bromo-2-chlorophenylamino)-5,6-dichloronicotinic acid (14.7 g,
37.0 mmol) in EtOH (300 mL) was slowly added trimethylsilyldiazomethane (60 mL,
S8

120 mmol, 2.0 M solution in hexanes). After the addition was complete the resulting
slurry was diluted with hexanes (300 mL) and the precipitates were filtered and washed
with hexanes to give 10.06 g of the desired product. The organic washes were
concentrated under reduced pressure to give the crude material that was purified by silica
gel flash column chromatography (CH2Cl2) to afford an additional 3.83 g of the desired
product. A total of 13.89 g (91%) of the desired product was isolated. MS APCI (+) m/z
1
409, 411, 413 (M+; Cl, Br pattern) detected. H NMR (400 MHz, DMSO-d6) 9.30 (s,
1H), 8.57 (s, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 2.4, 8.4 Hz, 1H), 7.05 (d, J = 8.4
Hz, 1H), 3.68 (s, 3H).
Step F: Preparation of methyl 4-(4-bromo-2-chlorophenylamino)-5-chloro-6-
methoxynicotinate: To a solution of methyl 4-(4-bromo-2-chlorophenylamino)-5,6-
dichloronicotinate (343 mg, 0.84 mmol) in a 4:1 mixture of MeOH-THF (5 mL) was
slowly added NaH (100 mg, 4.18 mmol) portionwise. The resulting mixture was stirred
for 16 h at room temperature. The reaction mixture was diluted with EtOAc, washed
with brine, dried over MgSO4, filtered, and concentrated under reduced pressure to give
the crude material that was purified by silica gel flash column chromatography (100%
CH2Cl2 to 10% MeOH in CH2Cl2) to afford 165 mg (49%) of the desired product. MS
1
APCI (-) m/z 403, 405, 407 (M-, Cl, Br pattern) detected. H NMR (400 MHz, CDCl3)
9.45 (s, 1H), 8.69 (s, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.26 (dd, J = 2.0, 8.4 Hz, 1H), 6.68
(d, J = 8.4 Hz, 1H), 4.08 (s, 3H), 3.91 (s, 3H).
Step G: Preparation of 4-(4-bromo-2-chlorophenylamino)-5-chloro-6-methoxynicotinic
acid:
To a suspension of methyl 4-(4-bromo-2-chlorophenylamino)-5-chloro-6-
methoxynicotinate (17 mg, 0.042 mmol) in a 1:1 mixture of THF-H2O (4 mL) was added
1 N aq NaOH (0.21 mL, 0.21 mmol) at room temperature. After stirring for 3 h, the
reaction mixture was concentrated under reduced pressure to half of the volume and
acidified to pH 1 with 1 N aq HCl. The resulting suspension was extracted with EtOAc-
THF, dried over MgSO4, filtered, and concentrated under reduced pressure to afford the
desired product quantitatively that was used directly without further purification. 1H
NMR (400 MHz, CD3OD) 8.70 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 2.4, 8.8
Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 4.03 (s, 3H).
S9

Step
H:
Preparation
of
4-(4-bromo-2-chlorophenylamino)-5-chloro-N-(2-
hydroxyethoxy)-6-methoxynicotinamide (14): The title compound 14 was prepared in
47% yield by the procedures previously described for compound 10 using 4-(4-bromo-2-
chlorophenylamino)-5-chloro-6-methoxynicotinic acid (15 mg, 0.038 mmol) and O-(2-
vinyloxyethyl)hydroxylamine (5.9 mg, 0.057 mmol). MS APCI (-) m/z 448, 450, 452
1
(M-, Cl, Br pattern) detected. H NMR (400 MHz, CD3OD) 8.17 (s, 1H), 7.59 (d, J = 2
Hz, 1H), 7.33 (dd, J = 8.4, 2.0 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 4.03 (s, 3H), 3.86 (t, J =
4.8 Hz, 2H), 3.68 (t, J = 4.8 Hz, 2H).
4-(4-Bromo-2-chlorophenylamino)-5,6-dichloro-N-(2-hydroxyethoxy)nicotinamide
(15).
The title compound 15 was prepared in 32 % yield by the procedures previously
described
for
compound
10
using
4-(4-bromo-2-chlorophenylamino)-5,6-
dichloronicotinic acid (35 mg, 0.088 mmol, prepared in step D for compound 14) and O-
(2-vinyloxyethyl)hydroxylamine (14 mg, 0.13 mmol). MS APCI (+) m/z 454, 456, 458
(M+, Cl, Br pattern) detected; 1H NMR (400 MHz, CD3OD) 8.26 (s, 1H), 7.63 (s, 1H),
7.40 (dd, J = 8.4, 2.4 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 3.82 (m, 2H), 3.70 (m, 2H).
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-N-hydroxy-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (20).
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-N-hydroxy-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide was prepared from 4-(4-bromo-2-fluorophenylamino)-N-
tert-butoxy-5-fluoro-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide
by
TFA
mediated deprotection. 4-(4-Bromo-2-fluorophenylamino)-N-tert-butoxy-5-fluoro-1-
methyl-6-oxo-1,6-dihydropyridine-3-carboxamide (35 mg, 0.081 mmol) was treated with
TFA (0.63 mL). After stirring for 4 days, the reaction mixture was concentrated under
reduced pressure to give the crude material that was purified by silica gel flash column
chromatography (10% MeOH in CH2Cl2) followed by sat’d aq NaHCO3 wash of an ethyl
acetate solution of the product to yield 10 mg (33%) of the desired product. MS APCI
1
(+) m/z 356, 358 (M-OH, Br pattern) detected; H NMR (400 MHz, CD3OD) δ 7.84 (s,
S10

1H), 7.35 (d, J = 10.4 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.01 (m, 1H), 3.57 (s, 3H); 19F
NMR (376 MHz, CD3OD) –126.0, –151.5.
The following compounds were prepared as described above.
4-(4-Bromo-2-fluorophenylamino)-5-chloro-N-(2-hydroxyethoxy)-1-methyl-6-oxo-
1,6-dihydropyridine-3-carboxamide (11).
1
MS APCI (+) m/z 434, 436 (M+, Cl, Br pattern) detected; H NMR (400 MHz, DMSO-
d6) 11.60 (s, 1H), 8.82 (s, 1H), 8.03 (s, 1H), 7.52 (d, J = 10.4 Hz, 1H), 7.28 (d, J = 8.4
Hz, 1H), 6.92 (dd, J = 8.4, 8.4 Hz, 1H), 4.69 (m, 1H), 3.68 (m, 2H), 3.52 (m, 2H), 3.47
(s, 3H); 19F NMR (376 MHz, DMSO-d6) –122.7.
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-N-(2-hydroxyethoxy)-1-methyl-6-oxo-
1,6-dihydropyridine-3-carboxamide (12).
1
MS APCI (+) m/z 418, 420 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD)
7.90 (s, 1H), 7.36 (dd, J = 10.4, 2.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.01 (td, J = 8.8,
4.0 Hz, 1H), 3.98 (m, 2H), 3.75 (m, 2H), 3.58 (s, 3H); 19F NMR (376 MHz, CD3OD) –
125.8, –152.1.
4-(4-Bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (13).
1
MS APCI (+) m/z 414, 416 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD)
7.93 (s, 1H), 7.33 (d, J = 10.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.65 (dd, J = 8.8, 8.8 Hz,
1H), 3.88 (m, 2H), 3.68 (m, 2H), 3.57 (s, 3H), 1.83 (s, 3H); 19F NMR (376 MHz,
CD3OD) –128.4; Anal. (C16H17BrFN3O4) C, H, N.
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-1-methyl-6-oxo-1,6-dihydropyridine-3-
carboxamide (16).
1
MS (+) m/z 358, 360 (M+, Br pattern) detected; H NMR (400 Hz, CD3OD) 8.11 (s,
1H), 7.36 (dd, J = 10.4, 2.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.02 (m, 1H), 3.58 (s, 3H);
19F NMR (376 MHz, CD3OD) –125.8, –152.3; Anal. (C13H10BrF2N3O2) H; C: calcd,
43.60; found, 42.98; N: calcd, 11.73; found, 11.04.
S11

4-(4-Bromo-2-fluorophenylamino)-5-fluoro-1-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylic acid (17).
1
MS APCI (-) m/z 357, 359 (M-1, Br pattern) detected; H NMR (400 MHz, CD3OD) δ
8.33 (s, 1H), 7.30 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 7.03 (td, J = 8.8, 4.0 Hz,
1H), 3.62 (s, 3H); 19F NMR (376 MHz, CD3OD) –125.2, –54.2.
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-N,1-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (18).
1
MS APCI (+) m/z 372, 374 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD) δ
7.93 (s, 1H), 7.35 (d, J = 10.4 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.0 (td, J = 8.8, 4.4 Hz,
1H), 3.57 (s, 3H), 2.83 (s, 3H); 19F NMR (376 MHz, CD3OD) –126.0, –152.1.
4-(4-Bromo-2-fluorophenylamino)-N-(cyclopropylmethyl)-5-fluoro-1-methyl-6-oxo-
1,6-dihydropyridine-3-carboxamide (19).
1
MS APCI (+) m/z 412, 414 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD) δ
7.97 (s, 1H), 7.32 (dd, J = 10.4, 1.6 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 6.98 (dt, J = 8.8,
4.8 Hz, 1H), 3.60 (s, 3H), 3.15 (d, J = 7.2 Hz, 2H), 1.04 (m, 1H), 0.54 (m, 2H), 0.26 (m,
2H); 19F NMR (376 MHz, CD3OD) –125.5, –151.4.
4-(4-Bromo-2-fluorophenylamino)-5-fluoro-N-methoxy-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (21).
1
MS APCI (+) m/z 388, 390 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD) δ
7.87 (s, 1H), 7.35 (dd, J = 10.4, 2.0 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.01 (td, J = 8.8,
4.4 Hz, 1H), 3.75 (s, 3H), 3.58 (s, 3H); 19F NMR (376 MHz, CD3OD) –125.7, –151.8.
4-(4-Bromo-2-fluorophenylamino)-N-ethoxy-5-fluoro-1-methyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (22).
1
MS APCI (+) m/z 402, 404 (M+, Br pattern) detected; H NMR (400 MHz, CD3OD) δ
7.87 (s, 1H), 7.35 (dd, J = 10.0, 1.6 Hz, 1H), 7.27 (d, J = 10.0 Hz, 1H), 7.01 (dt, J = 8.0,
19
4.0 Hz, 1H), 3.94 (q, J = 7.2 Hz, 2H), 3.58 (s, 3H), 1.27 (t, J = 6.8 Hz, 3H); F NMR
(376 MHz, CD3OD) –125.9, –152.0; Anal. (C15H14BrF2N3O3) C, H, N.
S12

4-(4-Bromo-2-fluorophenylamino)-N-methoxy-1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (23).
1
MS APCI (-) m/z 382, 384 (M-, Br pattern) detected; H NMR (400 MHz, CD3OD)
7.90 (s, 1H), 7.33 (m, 1H), 7.20 (m, 1H), 6.66 (dd, J = 8.8, 8.8 Hz, 1H), 3.65 (s, 3H), 3.57
19
(s, 3H), 1.83 (s, 3H); F NMR (376 MHz, CD3OD) –128.27; Anal. (C15H15BrFN3O3)
C, H, N.
4-(4-bromo-2-fluorophenylamino)-N-ethoxy-1,5-dimethyl-6-oxo-1,6-
dihydropyridine-3-carboxamide (24).
1
MS APCI (-) m/z 396, 398 (M-, Br pattern) detected; H NMR (400 MHz, CD3OD)
7.90 (s, 1H), 7.33 (dd, J = 10.8, 2.4 Hz, 1H), 7.20 (dd, J = 8.8, 1.6 Hz 1H), 6.65 (dd, J =
8.8, 8.8 Hz, 1H), 3.83 (q, J = 6.8 Hz, 2H), 3.57 (s, 3H), 1.83 (s, 3H), 1.22 (t, J = 6.8 Hz,
3H); 19F NMR (376 MHz, CD3OD) –128.32; Anal. (C16H17BrFN3O3) C, H, N.
S13

Elemental Analyses for Compounds for 13, 16, 22, 23, and 24
Compound
13
Formula
Calculated
Found
C16H17BrFN3O4
C 46.39
C 46.54
H
4.14
H
3.80
N 10.14
C 43.60
N 10.17
C 42.98
C13H10BrF2N3O2
C15H14BrF2N3O3
C15H15BrFN3O3
C16H17BrFN3O3
16
22
23
24
H
2.81
H
3.19
N 11.73
C 44.79
N 11.04
C 44.69
H
3.51
H
3.26
N 10.45
C 46.89
N 10.19
C 47.20
H
3.94
H
3.63
N 10.94
C 48.26
N 11.00
C 48.23
H
4.30
H
3.98
N 10.55
N 10.57
S14

Biological Assays
MEK enzyme assay: N-terminal 6 His-tagged, constitutively active MEK-1 (2-393) is
expressed in E. coli and protein is purified by conventional methods (Mansour S.J.;
Matten W.T.; Hermann A.S.; Candia J.M.; Rong S.; Fukasawa K.; Vande Woude G.F.;
Ahn N.G. Science 1994, 265, 966-970). The activity of MEK1 is assessed by measuring
the incorporation of γ-33P-phosphate from γ-33P-ATP onto N-terminal His tagged ERK2,
which is expressed in E. coli and is purified by conventional methods, in the presence of
MEK-1. The assay is carried out in 96-well polypropylene plate. The incubation mixture
(100 µL) comprises of 25 mM Hepes, pH 7.4, 10 mM MgCl2, 5 mM β-
glycerolphosphate, 100 µM Na-orthovanadate, 5 mM DTT, 5 nM MEK1, and 1 µM
ERK2. Inhibitors are suspended in DMSO, and all reactions, including controls are
performed at a final concentration of 1% DMSO. Reactions are initiated by the addition
of 10 µM ATP (with 0.5 µCi γ-33P-ATP/well) and incubated at ambient temperature for
45 minutes. Equal volume of 25% TCA is added to stop the reaction and precipitate the
proteins. Precipitated proteins are trapped onto glass fiber B filterplates, and excess
labeled ATP washed off using a Tomtec MACH III harvestor. Plates are allowed to air-
dry prior to adding 30 µL/well of Packard Microscint 20, and plates are counted using a
Packard TopCount.
pERK measurement in Malme-3M cells: Malme-3M melanoma cells were plated in
°
96-wells and treated with various concentrations of test compounds for 1 hr at 37 C. The
cells were fixed, permeabilized, and incubated with an anti-phospho-ERK antibody and
an anti-ERK1,2 antibody. Plates were washed and fluorescently-labeled secondary
antibodies were added. Plates were analyzed on a LICOR fluorescence imager. The
pERK signal is normalized to the total ERK signal.
Cell Viability assay: Malme-3M melanoma cells were plated in 96-wells and incubated
with various concentrations of test compounds. On Day 3, the number of viable cells was
measured using the MTS assay.
S15

Selectivity Data
The activity of analogs 13, 22, 23 and 24 against a number of important kinases was
evaluated at Upstate (Dundee). Compounds were tested at 10 uM.
Upstate Panel - % Inhibition @ 10 uM
Enzyme
Cmpd 13
6
Cmpd 22
Cmpd 23
Cmpd 24
MAPK2/ERK2
JNK/SAPK1c
SAPK2a/p38
SAPK2b/p38β2
MAPKAP-K1a
MAPKAP-K2
MSK1
7
15
10
9
-9
7
6
23
-3
13
3
23
20
16
-9
3
5
6
-8
1
8
15
7
14
5
2
6
PRAK
16
15
-8
18
14
-1
6
9
18
-1
3
PKA(rec)
PBK
-7
-4
0
PKCa
-3
7
PDK1
-7
-2
6
-9
12
-39
-2
12
10
-4
-3
-4
-1
8
-3
6
PKBa
-4
SGK
-15
18
17
13
19
11
-1
-18
13
21
4
-44
-1
15
13
2
p70 S6K
GSK3b
ROCK-II
AMPK
8
CHK1
13
4
8
CK2
4
LCK
13
9
19
25
12
9
CDK2/cyclin A
DYRK1a
20
15
10
2
S16

Non-Competitive Inhibition of 16
AR00340241-01
caMEK(withrespect toATP)
1.2
1.0
0.8
0.6
0.4
0.2
0.10
-0.05
0.00
0.05
0.10
0.15
1/[ATP] (µM)
nM
I =0
I =2.5
I =5
I =10
I =20
Vmax = 9.3
Km = 14
Ki = 6.6
To assess whether inhibitor 16 is a competitive inhibitor with respect to ATP, increasing
concentrations of inhibitor 16 (0 – 40 nM) was tested against constitutively active MEK1
in the presence of ATP that ranged from 6.67 to 66.7 µM (γ-33P-ATP added as tracer) and
1 µM ERK2. Reactions and method are as described. Data is analyzed using SigmaPlot.
In this plot, y-intercept = 1/Vmax and x-intercept = 1/Km. With increasing
concentrations of inhibitor 16, the Km for ATP remains unchanged (note the convergence
on the x-axis) while the Vmax of the MEK1 reaction decreases (different y-intercepts
with increasing [Inhibitor 16]). These results are characteristic of a noncompetitive
inhibitor.
S17

Hepatocyte Assay Methods
In vitro stability in the presence of hepatocytes was conducted as follows. Fresh or
cryopreserved hepatocytes were thawed if necessary, isolated from shipping media
and diluted to a viable cell density of 2 x 106 cells/mL according to the supplier’s
guidelines using Krebs-Henseleit buffer (KHB, pH 7.3, Sigma) supplemented with
amikacin (84 µg/mL), calcium chloride (1 mM), gentamicin (84 µg/mL), HEPES
(20 mM), heptanoic acid (4.2 µM) and sodium bicarbonate (2.2 mg/mL). Viability
was determined by trypan blue exclusion using a hemacytometer (3500 Hausser,
VWR). The 10 mM stock solution of test compounds in DMSO was diluted to 2 µM
using supplemented KHB buffer to create the working standard. A 50-µL aliquot of
test compound or control was added to each test well of a 96-well polypropylene plate
(Costar) immediately followed by the addition of 50 µL of the hepatocyte suspension.
One incubation plate was prepared for each timepoint with samples being prepared in
duplicate. Incubations were conducted at 37 °C, 5% CO2 and 100% relative humidity
in an incubator (Model 2300, VWR). At each timepoint, one incubation plate was
removed from the incubator, and a solution containing internal standard (100 µL,
2 µM labetalol) was added to each well. The plate was mixed at 700 rpm for 2
minutes on a plate shaker (IKA MTS 2/4 Digital Microtiter Shaker, VWR) and
immediately spun in a centrifuge at 2,000 x g for 10 minutes using an Allegra
benchtop centrifuge (Beckman Coulter). A 150-µL aliquot of the supernatant was
transferred from each well to a 96-well shallow plate (Costar). The plates were sealed
using reusable plate mats.
The relative concentrations of test compounds were quantitated using an LC-MS/MS
system comprised of an HTS-PAL autosampler (Leap Technologies), an HP1100
HPLC (Agilent), and an API4000 triple quadrupole mass spectrometer (Sciex).
Chromatographic separation of the analyte and internal standard was achieved using
an Aqua C18 column (72 x 2 mm, 3 µm particle size, Phenomenex) in conjunction
with gradient conditions using mobile phases A (aqueous 0.1% formic acid
containing 1% isopropanol) and B (0.1% formic acid in acetonitrile containing 1%
isopropanol). The total run time, including re-equilibration time, for a single injection
was 6 minutes. Mass spectrometric detection of the analytes was accomplished using
ESI+ ionization mode. Analyte responses were measured by multiple reaction
monitoring (MRM) of transitions unique to each compound.
Data were acquired and peak areas were calculated for test compounds and the
internal standard using the Analyst 1.4.1 software (Sciex). Peak area tables were
exported to Excel 2000, where the average analyte-to-internal standard peak area
ratios were used to calculate percent remaining (%rem), half-life (t1/2), predicted
intrinsic hepatic clearance (CLint), predicted hepatic clearance (CLh) and predicted
hepatic extraction ratio (ER).
S18

Caco-2 Methods
Upon receiving the plated Caco-2 cells, differentiation media was added to each transwell
and well of the 24-well plate. The Caco-2 plates are incubated overnight at 37 °C and 5%
CO2 with saturating humidity. The pH of the transport buffer was adjusted to 6.5 or 7.4,
using 1 M HCL for the apical and basolateral sides, respectively, and warmed to 37 °C in
a water bath. Dosing solutions were prepared using the apical and basolateral transport
media at a concentration of 10 µM for test compounds and containing 25 µM lucifer
yellow to monitor monolayer integrity.
Prior to conducting the assay, to ensure the integrity of Caco-2 monolayers, trans
epithelial electrical resistance (TEER) was measured using a Millipore Millicell-ERS
TEER device. A reading of at least 1,000 from the TEER device indicated that the
cells were acceptable for the permeability assay.
Additionally, the Caco-2 cultures were considered acceptable for transport studies if the
following criteria were met:
• A low mannitol permeability transport rate (Papp) of 20 nm/s.
• A propranolol:mannitol permeability ratio of 5.
• A vinblastine with verapamil:vinblastine permeability ratio of 1.5.
The typical experimental conditions were as follows (conducted at In Vitro Technologies
on a Caco-2 plate from the same culture):
1. Mannitol permeability: A
approximately 152 nM and 4 µCi/mL in a 2-hour incubation.
B transport was measured using [3H]mannitol at
2. Propranolol permeability: A
B transport was measured using [3H]propranolol
at approximately 190 nM and 4 µCi/mL in a 2-hour incubation.
3. Vinblastime permeability: A
B transport was measured using [3H]vinblastine
at approximately 44 nM and 0.5 µCi/mL in a 2-hour incubation.
4. Vinblastine permeability in the presence of verapamil: A
B transport was
measured using [3H]vinblastine at approximately 22 nM and 0.25 µCi/mL in a
solution containing 100 µM of verapamil.
Verapamil and progesterone, both high permeability compounds, metolazone, a medium
permeability compound, and erythromycin, a low permeability compound, were used as
controls in the assay. Stock solutions of each of these controls, as well as ARRY-334543
were prepared at 10 mM in DMSO. Compounds were diluted 1:1000 to 10 µM using the
appropriate transport buffer.
For the apical to basolateral determination, 100 µL of the test compound in apical
transport buffer was added to the apical side of the individual transwells and 600 µL of
S19

basolateral media was added to each well. For the basolateral to apical determination,
600 µL of test compound in basolateral transport buffer was added to each well and 100
µL apical transport buffer added to each transwell. All compounds were tested in
duplicate wells to assess transport in both directions. The plates were incubated for 2
hours on a Lab-Line Instruments Titer Orbital Shaker (VWR, Cat No. 57019-600) at 50
rpm and 37°C with 5%CO2. Plates were removed from the incubator and 75 µL of media
is removed from the apical and basolateral portion of each well and added to 75 µL of
internal standard (4 µM labetalol) in acetonitrile.
The plates were first read using a Molecular Devices Gemini Fluorometer to evaluate
lucifer yellow concentrations at an excitation/emission wavelength of 425/535 nm.
Lucifer yellow transport values were below 1% for apical to basolateral and below 2%
for basolateral to apical when compared to the dosing solutions. The plates were sealed
with reusable plate mats and the contents of each well analyzed by LC-MS/MS. The
compound concentrations were determined from the ratio of the peak areas of the
compound to the internal standard (labetalol) in comparison to the dosing solution.
Instrumentation comprised of a CTC PAL autosampler coupled with a Finnigan Surveyor
LC system. Mass selective detection was achieved using a Finnigan LCQ Deca ion trap
mass spectrometer. When performing LC/MS analysis of test compounds, 10-20 L of
the sample from each well was injected for analysis. Separations were achieved using a
XTerra C18 column (2.1 or 3.0 x 30mm, 3.5 µm particle size, Waters) and a 0.4 mL/min.
linear gradient. The gradient was from 95% aqueous buffer (formic or NH4OH) to 95%
acetonitrile over 4-5 minutes. An optimized, dedicated MS/MS method was created by
maximizing the MS/MS fragment ion signal during infusion of the stock solutions into a
mobile phase equivalent to that present midway through the HPLC gradient.
S20

Aqueous Solubility Assay
The thermodynamic aqueous solubility of compounds was measured using a modified
shake-flask method. Crystallinity of each compound was confirmed using a polarizing
light microscope (Olympus BX51). For each compound, 0.5 mL of aqueous buffer (10
mM potassium phosphate), pH 6.5, was added to 0.5 mg of dry compound and the
mixture was swirled at 350 rpm at room temperature for 24 hours. Aliquots were
subsequently removed and filtered for HPLC analysis. Standards of known concentration
were also prepared and analyzed for each compound in order to create a calibration
curve. Analysis was accomplished using a HPLC/PDA system comprised of an Alliance
2795 Separations System (Waters) and a 2996 Photodiode Array Detector (Waters).
S21

Rodent Pharmacokinetics:
In vivo pharmacokinetic studies were performed in male Sprague Dawley rats (6-10
weeks of age) allowed food and water ad lib. Intravenous dose solutions were prepared
in 10% DMSO/90% normal saline (0.9% NaCl) at a concentration of 1 mg/mL to yield a
dose of 1 mg/kg at a dose volume of 1 mL/kg, IV. Oral dose solutions were prepared in
1% CMC/0.5% Tween 80 at a concentration of 2 mg/mL to yield a dose of 10 mg/kg at a
dose volume of 5 mL/kg, PO. Whole blood samples (in EDTA) were obtained vial the
tail vein at the following timepoints post dose administration:
IV: 1, 5, 15 and 30 min; 1, 2, 4, 8, 12 and 24 hrs
PO: 15 and 30 min; 1, 2, 4, 8, 12, 24, 48 and 72 hrs
Blood samples were centrifuged and resulting plasma was analyzed for compound
concentration using a LC/MS/MS method. The method utilized protein precipitation by
methanol of the plasma sample followed by centrifugation and detection of each
compound using reversed phase liquid chromatography with triple quadrapole MS/MS
selective detection. Labetalol was used as the internal standard. The method had a
validated quantitation range of 9 ng/mL to 20 µg/mL. Higher sample concentrations were
quantitated by diluting samples by up to 10-fold using plasma from naïve animals.
Pharmacokinetic (PK) parameters were calculated by established non-compartmental
methods using WinNonlin Pharsight, Inc).
S22

PK / PD Assay
Tumor growth and harvest. HT-29 cells (ATCC # HTB-38) were grown as monolayer
cultures at 37oC in a humidified tissue culture incubator with a 5% CO2 atmosphere.
Growth medium was McCoy’s 5A (ATCC #30-2007) with 10% FBS (ATCC #30-2020),
GlutaMAX (Gibco #35050-061) and antibiotic-antimycotic (Gibco #15240-062). Cells
were harvested at approximately 80% confluency using 0.05% trypsin/EDTA (Cellgro
#25-052-C1), pelleted, and resuspended at 3x107/ml in PBS. 100 ul of cell suspension
(3x106 cells) was implanted subcutaneously in the right flank of female athymic Ncr-
nu/nu mice (Taconic – NCI stock, 6 – 10 weeks of age). 10 - 14 days after implantation,
tumor volume reached at average of 250-350 mm3 (volume determined by caliper
measurement, using the formula volume = length x width2 / 2). Mice with tumors of the
appropriate size were selected and randomized into groups (n=4 per group) of similar
average volume.
Compounds were administered by oral gavage, at a volume of 10 ml/kg, in a dosing
vehicle containing 1% methyl cellulose and 1% Tween-80. At the appropriate timepoints
after compound administration, mice were euthanized by CO2 inhalation and tumor and
blood collected for analysis. Blood was collected by cardiac puncture, using sodium
citrate as an anticoagulant, and plasma collected and stored at –20oC until analysis.
Quantitation of compound in plasma was carried out as described in the rodent PK
section. Tumors were excised, flash frozen in liquid nitrogen, and stored at –80oC until
analysis for levels of phospho-ERK and total ERK.
Analysis of phospho-ERK and total ERK in tumor lysates. All steps in preparation of
tumor lysate were carried out at 4oC. Tumor fragments were mixed with an equal volume
of lysis buffer (50mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid,
0.05% SDS, 2mM EDTA) with 1% of each of the following phosphatase and protease
inhibitors: Sigma #P8340, Sigma # P2850, Sigma #P5726. Samples were homogenized
using a Tissue Tearor Model 398 tissue homogenizer (Biospec Products). The
homogenized lysate was centrifuged for 10 min at 18,000 x g and the supernatant
S23

collected. Protein concentration of the lysate supernatant was determined using the
Comassie Plus protein assay (Pierce #1856210). Lysate was stored at –80oC
Samples for gel analysis contained the volume of lysate supernatant needed for 150ug of
protein diluted to 65ul with lysis buffer, plus 25ul of 4X gel loading buffer (NuPAGE
LDS sample buffer, Invitrogen #NP0007) and 10ul of beta-mercaptoethanol. Samples
were heated to 65oC for 10 min prior to loading onto gels.
Fifteen ul of the above samples (containing 22.5 ug of total protein) were loaded onto 12-
well NuPAGE precast 10% bis-tris gels (Invitrogen #NP0302). Electrophoresis was
carried out in a NOVEX X-cell gell apparatus (Invitrogen), using conditions and buffers
recommended by the supplier. After completion of electrophoresis, proteins were
transferred to nitrocellulose membrane using a NOVEX transblot apparatus, using
conditions and buffers recommended by the supplier.
After completion of electrophoresis, nitrocellulose membranes were washed 2X in TBST
pH 8.0, followed by blocking in Odyssey blocking buffer (Licor Biosciences #927-
40000) at room temperature for 1h or overnight at 4oC. Membranes were then
incubated with phospho-p44/42 MAPK (thr202/tyr204) antibody (Cell Signaling
Technology #9101) diluted 1:1000 in Odyssey blocking buffer with 0.1% Tween-20, for
either 1h at room temperature or 4oC overnight. Membranes were then washed 3X with
TBST, followed by incubation with Alexa-fluor 680 conjugated goat anti-rabbit IgG
(Molecular Probes #A-21109), diluted 1:2000 as above, for 1h at room temperature.
Membranes were then washed 2X with TBST, followed by a final wash with TBS pH
8.0.
Membrane images were captured using a Licor Odyssey imaging system (Licor
Biosciences), with image capture on the 700 nm channel. Quantitation of band intensity
was carried out using Odyssey version 1.2 software (Licor Biosciences).
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Total ERK was quantitated on the same membranes. Dilutions and washes were as
described above, using ERK-1 rabbit polyclonal IgG (Santa Cruz Biotechnology #K-23),
IRdye 800 conjugated affinity purified goat anti-rabbit IgG (Rockland Immunochemicals
#611-132-122), and Licor image capture on the 800 nm channel.
Data analysis. Each gel contained vehicle control samples. The phospho-ERK / total
ERK signal ratio was calculated for each sample. These values were then normalized to
the average vehicle control value for that gel, and reported as % of control value (mean
and standard error for each treatment group and timepoint).
S25