6830 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Das et al.
8.8 Hz, 2H), 5.21 (d, J ) 13.7 Hz, 1H), 4.44 (d, J ) 13.7 Hz, 1H),
3.71 (s, 3H), 2.45 (s, 3H), 1.72 (s, 3H). MS (ESI) m/z 515 (M +
H)+.
1H), 7.39 (dd, J ) 1.65, 7.38 Hz, 1H), 7.29-7.24 (m, 2H), 6.29
(s, 1H), 3.70-3.68 (m, 4H), 3.53-3.51 (m, 4H), 2.23 (s, 3H).
HRMS (ESI) m/z calcd for C19H20ClN6O2S (M + H)+, 431.1057;
found, 431.1055. Anal. Calcd for C19H19ClN6O2S‚1.0HCl: C, 48.83;
H, 4.31; N, 17.98. Found: C, 48.76; H, 4.21; N, 17.80. Purity:
>98% (method A; tR ) 3.44 min).
N-(2-Chloro-6-methylphenyl)-2-(2-methyl-6-morpholinopyri-
midin-4-ylamino)-1,3-thiazole-5-carboxamide (12t): 1H NMR
(400 MHz, DMSO-d6) δ 11.52 (s, 1H), 9.88 (s, 1H), 8.21 (s, 1H),
7.39 (d, J ) 6.10 Hz, 1H), 7.29-7.23 (m, 3H), 6.04 (s, 1H), 3.67-
3.65 (m, 4H), 3.49-3.46 (m, 4H), 2.41 (s, 3H), 2.23 (s, 3H). HRMS
(ESI) m/z calcd for C20H22ClN6O2S (M + H)+, 445.1213; found,
445.1234. Purity: 95% (method A; tR ) 3.34 min).
2-(6-Chloro-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-meth-
ylphenyl)-1,3-thiazole-5-carboxamide (17): A solution of 16 (6.6
g, 13 mmol) dissolved in a mixture of CH2Cl2 and TFA (50 mL,
1:1) was treated with triflic acid (6.8 g, 45 mmol) and stirred at rt
for 3 h. The mixture was poured onto crushed ice (150 g) and
extracted with CHCl3 (100 mL, 3×). The desired product precipi-
tated from solution upon standing at room temperature and was
collected by filtration and dried in vacuo to give compound 17 (5.0
g, 99%). 1H NMR (400 MHz, DMSO-d6) δ 12.24 (br s, 1H), 10.02
(s, 1H), 8.32 (s, 1H), 7.41 (d, J ) 7.7 Hz, 1H), 7.30 (dd, J ) 6.1,
6.7 Hz, 1H), 7.26 (d, J ) 7.7 Hz, 1H), 6.95 (s, 1H), 2.59 (s, 3H),
2.24 (s, 3H). MS (ESI) m/z 395 (M + H)+.
N-(2-Chloro-6-methylphenyl)-2-(2-methyl-6-(3-morpholino-
propylamino)pyrimidin-4-ylamino)-1,3-thiazole-5-carboxam-
N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-pip-
erazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-car-
boxamide, Hydrochloride Salt (2): A mixture of 17 (34.7 g, 88.1
mmol), diisopropylethylamine (3.6 mL, 0.17 mol), and 1-(2-
hydroxyethyl)piperazine (54 mL, 0.44 mol) in 1,4-dioxane (300
mL) was refluxed for 12 h. The mixture was concentrated under
vacuum, and the solid was triturated successively with H2O, aqueous
MeOH, and Et2O (2×) and dried in vacuo. The solid was
resuspended in ether-methanol, treated with 2 N HCl (44 mL),
and vigorously stirred for 30 min. The precipitate was collected
by filtration and triturated successively with ether, ether-methanol,
and then ether. The solid was dried in vacuo to give 2 (42.2 g,
91%) as its hydrochloride salt. Mp ) 279-280 °C (free base). 1H
NMR (400 MHz, DMSO-d6) δ 11.67 (br s, 1H), 10.50 (br s, 1H),
9.96 (s, 1H), 8.27 (s, 1H), 7.40 (d, J ) 7.7 Hz, 1H), 7.28 (dd, J )
6.6, 6.7 Hz, 1H), 7.25 (d, J ) 7.7 H, 1 Hz), 6.17 (s, 1H), 4.33 (d,
J ) 12.6 Hz, 2H), 3.79 (dd, J ) 5.0, 5.5 Hz, 2H), 3.60 (d, J )
11.6 Hz, 2H), 3.38 (dd, J ) 12.1, 12.6 Hz, 2H), 3.22 - 3.19 (m,
2H), 3.13 - 3.07 (m, 2H), 2.45 (s, 3H), 2.24 (s, 3H). 13C NMR
(125 MHz, DMSO-d6) δ 165.7, 162.8, 162.1, 160.4, 157.5, 141.2,
139.4, 133.8, 133.0, 129.6, 128.8, 127.6, 126.5, 84.0, 58.1, 55.2,
51.1 (2), 41.2 (2), 25.7, 18.8. HRMS (ESI) m/z calcd for C22H27-
ClN7O2S (M + H)+, 488.1635; found, 488.1636. Anal. Calcd for
C22H26ClN7O2S: C, 54.14; H, 5.37; N, 20.09. Found: C, 53.90;
H, 5.30; N, 20.07. Purity: 100% (method A; tR ) 2.72 min).
N-(2-Chloro-6-methylphenyl)-2-(6-(2-hydroxyethylamino)py-
ridin-2-ylamino)-1,3-thiazole-5-carboxamide (12p): 1H NMR
(400 MHz, DMSO-d6) δ 11.27 (s, 1H), 9.77 (s, 1H), 8.19 (s, 1H),
7.38 (dd, J ) 2.17, 7.23 Hz, 1H), 7.32-7.22 (m, 3H), 6.16 (d, J )
7.63 Hz, 1H), 6.09 (d, J ) 8.13 Hz, 1H), 3.60-3.57 (m, 2H), 3.50-
3.45 (m, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C18H19-
ClN5O2S (M + H)+, 404.0948; found, 404.0964. Anal. Calcd for
C18H18ClN5O2S‚1.0HCl‚0.2H2O: C, 48.69; H, 4.40; N, 15.77.
Found: C, 48.65; H, 4.18; N, 15.59. Purity: >99% (method A; tR
) 3.08 min).
N-(2-Chloro-6-methylphenyl)-2-(6-(3-morpholinopropylami-
no)pyridin-2-ylamino)-1,3-thiazole-5-carboxamide (12q): 1H NMR
(400 MHz, DMSO-d6) δ 11.39 (s, 1H), 9.55 (s, 1H), 9.57 (br s,
1H), 8.27 (s, 1H), 7.39 (d, J ) 7.63 Hz, 1H), 7.33 (dd, J ) 7.63
Hz, 7.63 Hz, 1H), 7.29-7.22 (m, 2H), 6.97 (br s, 1H), 6.19 (d, J
) 7.63 Hz, 1H), 6.05 (d, J ) 8.13 Hz, 1H), 3.88 (d, J ) 13.74 Hz,
3H), 3.55 (t, J ) 11.69 Hz, 2H), 3.46-3.37 (m, 3H), 3.29-3.23
(m, 2H), 3.03-2.93 (m, 2H), 2.23 (s, 3H), 2.03-1.97 (m, 2H).
HRMS (ESI) m/z calcd for C23H28ClN6O2S (M + H)+, 487.1683;
found, 487.1696. Purity: >98% (method A; tR ) 2.87 min).
2-(6-(3-(1H-Imidazol-1-yl)propylamino)pyridin-2-ylamino)-
N-(2-chloro-6-methylphenyl)-1,3-thiazole-5-carboxamide (12r):
1H NMR (500 MHz, CD3OD) δ 8.99 (s, 1H), 8.36 (s, 1H), 7.70 (s,
1H), 7.65 (br s, 1H), 7.54 (s, 1H),7.36-7.38 (dd, J ) 2.2, 7.2 Hz,
1H), 7.25-7.28 (m, 2H), 6.39-6.44 (m, 2H), 4.46 (t, J ) 7.4 Hz,
2H), 3.55 (t, J ) 7.2 Hz, 2H), 2.32-2.36 (m, 5H). HRMS (ESI)
m/z calcd for C22H23ClN7OS (M + H)+, 468.1373; found, 468.1394.
Purity: >98% (method A; tR ) 2.83 min).
1
ide (12u): H NMR (500 MHz, DMSO-d6): δ 11.16 (br s, 1H),
10.08 (s, 1H), 8.33 (s, 1H), 7.40 (dd, J ) 1.65, 7.38 Hz, 1H), 7.30-
7.25 (m, 3H), 6.28 (br s, 1H), 4.01-3.90 (m, 2H), 3.89-3.80 (m,
2H), 3.53-3.43 (m, 2H), 3.19-3.13 (m, 2H), 3.09-3.03 (m, 2H),
2.60-2.53 (m, 2H), 2.45 (s, 3H), 2.24 (s, 3H), 2.07-1.99 (m, 2H).
HRMS (ESI) m/z calcd for C23H29ClN7O2S (M + H)+, 502.1792;
found, 502.1787. Purity: >98% (method A; tR ) 2.25 min).
N-(2-Chloro-6-methylphenyl)-2-(6-(2-hydroxyethylamino)-2-
methylpyrimidin-4-ylamino)-1,3-thiazole-5-carboxamide (12v):
1H NMR (400 MHz, DMSO-d6): δ 11.27 (s, 1H), 9.77 (s, 1H),
8.19 (s, 1H), 7.38 (dd, J ) 2.17, 7.23 Hz, 1H), 7.32-7.22 (m,
3H), 6.16 (d, J ) 7.63 Hz, 1H), 6.09 (d, J ) 8.13 Hz, 1H), 3.60-
3.57 (m, 2H), 3.50-3.45 (m, 2H), 2.23 (s, 3H). HRMS (ESI) m/z
calcd for C18H20ClN6O2S (M + H)+, 419.1057; found, 419.1047.
Purity: 95% (method A; tR ) 2.68 min).
N-(2-Chloro-6-methylphenyl)-2-(6-(4-(hydroxymethyl)piperi-
din-1-yl)-2-methylpyrimidin-4-ylamino)-1,3-thiazole-5-carboxa-
1
mide (12w): H NMR (500 MHz, CD3OD) δ 8.22 (s, 1H), 7.35
(dd, J ) 7.1, 1.6 Hz, 1H), 7.26-7.23 (m, 2H), 6.39 (br s, 1H),
3.45 (d, J ) 6.0 Hz, 2H), 3.31-3.29 (m, 4H), 2.65 (s, 3H), 2.31
(s, 3H), 2.00-1.80 (m, 3H), 1.35-1.32 (m, 2H). LC/MS (ESI):
m/z 473 (M + H)+. Purity: 98% (method A; tR ) 3.05 min).
Lck Enzyme Assay. For the preparation of recombinant Lck,
human Lck was prepared as a His-tagged fusion protein using the
commercially available (Life Technologies) baculovirus vector
pFastBac Hta in insect cells. A cDNA encoding human Lck isolated
by PCR was inserted into the vector, and the protein was expressed
using the methods described by the manufacturer. The Lck purified
by nickel affinity chromatography was incubated in kinase buffer
(20 mM MOPS, pH 7, 10 mM MgCl2) in the presence of the test
compound. The reaction was initiated by the addition of the
substrates to the final concentration of 1 µM ATP, 3.3 µCi/mL
[33P] g-ATP, and 0.1 mg/mL acid denatured enolase and was
stopped after 10 min by the addition of 10% trichloroacetic acid
and 100 mM sodium pyrophosphate, followed by 2 mg/mL bovine
serum albumin. The labeled enolase protein substrate was precipi-
tated at 4 °C, harvested onto Packard Unifilter plates, and counted
in a Topcount scintillation counter.
T-Cell Proliferation Assay. A 96-well plate was coated with a
monoclonal antibody to CD3 (G19-4), the antibody was allowed
to bind, and then the plate was washed. Normal human peripheral
blood T-cells were added to the wells, along with the test compound
and anti-CD28 antibody (E.3), to provide costimulation. After 3
days, the [3H]-thymidine was added to the cells, further incubation
occurred, and the cells were harvested and counted in a scintillation
counter to measure proliferation.
Inhibition of IL-2 Production Ex Vivo in Mice. BALB/c
female mice were dosed by oral gavage (po) with either vehicle
alone (propylene glycol-water, 1:1, v/v) or compound 12m
dissolved in vehicle. After 2 h, blood was collected into heparain
anticoagulant. Triplicate cultures of each sample containing 100
µL whole blood and anti-CD3 antibody (10 µg/mL), anti-CD28
antibody (10 µg/mL), and goat anti-hamster IgG antibody (30 µg/
mL) were established in 96-well plates. Plates were incubated for
24 h at 37 °C in 5% CO2. Culture supernatants were collected and
N-(2-Chloro-6-methylphenyl)-2-(6-morpholinopyrimidin-4-
ylamino)-1,3-thiazole-5-carboxamide (12s): 1H NMR (500 MHz,
DMSO-d6) δ 11.75 (br s, 1H), 9.98 (s, 1H), 8.45 (s, 1H), 8.26 (s,