Potent and Orally ActiVe Inhibitors of Lck
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 16 4989
+
HRMS (M + H] ) calcd: 585.29725 found: 585.29669. Anal.
1.46-1.54 (m, 4 H), 1.78-1.87 (m, 2 H), 2.09 (s, 6 H), 2.28-
2.41 (m, 6 H), 3.75 (s, 3 H), 3.85-3.91 (m, 5 H), 6.51 (s, 1 H),
6.54 (s, 1 H), 6.69 (dd, J ) 8.59, 2.53 Hz, 1 H), 6.82 (d, J ) 2.53
Hz, 1 H), 7.04-7.12 (m, 5 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.47 (d,
J ) 5.81 Hz, 1 H), 8.34 (d, J ) 5.81 Hz, 1 H), 9.34 (s, 1 H).
(C
36
H
36
N
6
O
2
2
‚0.9 H O): C, H, N.
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-(methyl-
2
oxy)-4-(4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)-
1
carbamate (33). H NMR (400 MHz, DMSO-d
H), 2.21 (s, 3 H), 2.38-2.47 (m, 4 H), 2.80-2.91 (m, 4 H), 3.66
s, 3 H), 3.75 (s, 3 H), 3.88 (s, 3 H), 6.31-6.39 (m, 1 H), 6.68 (dd,
J ) 8.72, 2.53 Hz, 1 H), 6.71-6.78 (m, 1 H), 6.79-6.85 (m, 2 H),
.03-7.12 (m, 3 H), 7.33 (d, J ) 8.59 Hz, 1 H), 7.45 (d, J ) 5.68
Hz, 1 H), 8.34 (d, J ) 5.81 Hz, 1 H), 9.23 (s, 1 H). HPLC purity:
6
) δ ppm 2.09 (s,
+
6
HRMS (M + H] ) calcd: 612.31805 found: 612.31809. Anal.
(
41 5 5
(C35H N O ): C, H, N.
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-(methyl-
7
oxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-
1
4-pyrimidinyl)carbamate (42). H NMR (400 MHz, DMSO-d
6
)
+
1
00%. HRMS (M + H] ) calcd: 599.29764 found: 599.29696.
,6-Dimethylphenyl 2-((3,5-bis(methyloxy)-4-(4-methyl-1-pip-
erazinyl)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)-
carbamate (34). 1H NMR (400 MHz, DMSO-d ) δ ppm 2.08 (s,
H), 2.20 (s, 3 H), 2.36 (s, 4 H), 2.95 (s, 4 H), 3.62 (s, 6 H), 3.78
s, 3 H), 3.81 (s, 3 H), 6.61 (dd, J ) 8.72, 2.40 Hz, 1 H), 6.72 (d,
δ ppm 1.78-1.88 (m, 2 H), 2.093 (s, 6 H), 2.24 (s, 3 H), 2.36-
2.47 (m, 6 H), 2.50 (m, 4 H), 3.65 (s, 3 H), 3.76 (s, 3 H), 3.83-
3.91 (m, 5 H), 6.40 (s, 1 H), 6.68 (dd, J ) 8.59, 2.53 Hz, 1 H),
6.76 (m, 1 H), 6.80 (d, J ) 2.53 Hz, 1 H), 6.84-6.92 (m, 1 H),
7.01-7.14 (m, 3 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.43 (d, J ) 5.81
Hz, 1 H), 8.35 (d, J ) 5.81 Hz, 1 H), 9.22 (s, 1 H). HPLC Purity:
2
6
6
(
+
J ) 2.40 Hz, 1 H), 6.89 (s, 2 H), 7.03-7.10 (m, 3 H), 7.15 (d, J
100%. HRMS (M + H] ) calcd: 657.33951 found: 657.33974.
)
1
5.81 Hz, 1 H), 7.33 (d, J ) 8.59 Hz, 1 H), 8.36 (d, J ) 5.68 Hz,
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrim-
+
H), 9.14 (s, 1 H). HPLC purity: 100%. HRMS (M + H] )
1
calcd: 629.3009 found: 629.30863.
idinyl)carbamate (44). H NMR (400 MHz, DMSO-d ) δ ppm
6
2
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
4-methyl-1-piperazinyl)phenyl)amino)-4-pyrimidinyl)carbam-
ate (35). H NMR (400 MHz, DMSO-d
1.79-1.88 (m, 2 H), 2.09 (s, 6 H), 2.15 (s, 3 H), 2.23-2.44 (m, 10
H), 3.76 (s, 3 H), 3.85 (s, 3 H), 3.92-3.98 (m, 2 H), 6.63-6.69
(m, 1 H), 6.70-6.77 (m, 1 H), 6.79 (d, J ) 2.53 Hz, 1 H), 6.87-
6.93 (m, 1 H), 7.04-7.14 (m, 4 H), 7.34 (d, J ) 8.46 Hz, 1 H),
7.51 (d, J ) 5.81 Hz, 1 H), 8.38 (d, J ) 5.81 Hz, 1 H), 9.51 (s, 1
(
1
6
) δ ppm 2.09 (s, 6 H),
2
.22-2.29 (m, 3 H), 2.86-2.93 (m, 4 H), 3.76 (s, 3 H), 3.85 (s, 3
H), 6.59-6.69 (m, 2 H), 6.80 (d, J ) 2.53 Hz, 1 H), 6.90 (d, J )
+
8
7
.34 Hz, 1 H), 6.99-7.12 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H),
H). HRMS (M + H] ) calcd: 645.31952 found: 645.31855. Anal.
.51 (d, J ) 5.81 Hz, 1 H), 8.38 (d, J ) 5.68 Hz, 1 H), 9.51 (s, 1
(C35
6 5 2
H41FN O ‚0.5 H O): C, H, N.
+
H). HRMS (M + H] ) calcd: 587.27766 found: 587.27708. Anal.
2,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((3-fluoro-4-
((3-(1-piperidinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl)car-
(C H35FN O
32 6 4
): C, H, N.
,6-Dimethylphenyl 2,4-Bis(methyloxy)phenyl(2-((4-((3-(dimeth-
ylamino)propyl)oxy)phenyl)amino)-4-pyrimidinyl)carbamate (37).
2
6
bamate (45). 1H NMR (400 MHz, DMSO-d ) δ ppm 1.34-1.42
(m, 2 H), 1.46-1.53 (m, 4 H), 1.79-1.87 (m, 2 H), 2.09 (s, 6 H),
2.28-2.40 (m, 6 H), 3.76 (s, 3 H), 3.85 (s, 3 H), 3.91-3.99 (m, 2
H), 6.66 (dd, J ) 8.72, 2.53 Hz, 1 H), 6.73 (t, J ) 9.35 Hz, 1 H),
6.79 (d, J ) 2.53 Hz, 1 H), 6.87-6.94 (m, 1 H), 7.02-7.14 (m, 4
H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.51 (d, J ) 5.81 Hz, 1 H), 8.38
1
H NMR (400 MHz, DMSO-d
.13 (m, 6 H), 2.17 (s, 6 H), 2.37 (t, J ) 7.07 Hz, 2 H), 3.77 (s, 3
H), 3.84-3.94 (m, 5 H), 6.54 (d, J ) 8.84 Hz, 2 H), 6.70 (dd, J )
.59, 2.53 Hz, 1 H), 6.83 (d, J ) 2.53 Hz, 1 H), 7.02-7.17 (m, 5
6
) δ ppm 1.75-1.89 (m, 2 H), 2.05-
2
8
+
H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.48 (d, J ) 5.81 Hz, 1 H), 8.35
(d, J ) 5.68 Hz, 1 H), 9.51 (s, 1 H). HRMS (M + H] ) calcd:
(
(
d, J ) 5.81 Hz, 1 H), 9.33 (s, 1 H). HPLC Purity: 96%. HRMS
630.30862 found: 630.30831. Anal. (C35
H, N.
5 5 2
H40FN O ‚0.25 H O): C,
+
M + H] ) calcd: 572.28675 found: 572.28597.
2
,6-Dimethylphenyl-2,4-bis(methyloxy)phenyl(2-((3-fluoro-4-
2,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((3-(4-methyl-
1-piperazinyl)propyl)-oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis-
(methyloxy)phenyl)carbamate (43). A resealable tube was charged
with 2,6-dimethylphenyl 2-chloropyrimidin-4-yl(2,4-dimethoxyphe-
nyl)carbamate (22a) (0.27 g, 0.55 mmol), 3,5-dimethoxy-4-(3-(4-
methylpiperazin-1-yl)propoxy)aniline (11g) (0.19 g, 0.65 mmol),
trifluoroacetic acid (0.29 g, 0.20 mL, 1.2 mmol), and 2-propanol
(3.0 mL). The system was flushed with argon, and the tube was
sealed. The mixture was heated at 100 °C for 18 h. The reaction
mixture was cooled to room temperature and concentrated to afford
a brown oil. The oil was diluted with ethyl acetate and partitioned
between ethyl acetate and saturated aqueous potassium carbonate.
The organic layer was separated, washed with brine, dried over
anhydrous sodium sulfate, and concentrated to afford 2,6-dimeth-
ylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)-pro-
pyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)-
(
(2-(4-methyl-1-piperazinyl)ethyl)oxy)phenyl)amino)-4-pyrim-
idinyl)carbamate (38). H NMR (400 MHz, DMSO-d
1
6
) δ ppm
2
.09 (s, 6 H), 2.16 (s, 3 H), 2.24-2.39 (m, 4 H), 2.66 (t, J ) 5.81
Hz, 3 H), 3.77 (s, 3 H), 3.84 (s, 3 H), 4.02 (t, J ) 5.81 Hz, 2 H),
6
1
.62-6.69 (m, 1 H), 6.72-6.81 (m, 2 H), 6.91 (d, J ) 10.11 Hz,
H), 7.02-7.16 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H), 7.50 (d, J
)
(
5.68 Hz, 1 H), 8.38 (d, J ) 5.68 Hz, 1 H), 9.52 (s, 1 H). HRMS
+
39
M + H] ) calcd: 631.30387 found: 631.30582. Anal. (C34H -
FN O
6 5
‚0.5 H
,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((2-(4-methyl-
-piperazinyl)ethyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(m-
2
O): C, H, N.
2
1
1
ethyloxy)phenyl)carbamate (39). H NMR (400 MHz, DMSO-
d
6
) δ ppm 2.07-2.12 (m, 6 H), 2.15 (s, 3 H), 2.19-2.42 (m, 8 H),
2
.57 (t, J ) 5.94 Hz, 2 H), 3.64 (s, 6 H), 3.79 (s, 3 H), 3.82 (s, 3
H), 3.85 (t, J ) 6.06 Hz, 2 H), 6.62 (dd, J ) 8.59, 2.53 Hz, 1 H),
1
6
.73 (d, J ) 2.53 Hz, 1 H), 6.94 (s, 2 H), 7.03-7.12 (m, 3 H),
carbamate (43) (0.189 g, 42%) as an off-white solid. H NMR (400
7
)
.14 (d, J ) 5.81 Hz, 1 H), 7.34 (d, J ) 8.59 Hz, 1 H), 8.37 (d, J
6
MHz, DMSO-d ) δ ppm 1.67-1.76 (m, 2 H), 2.10 (s, 6 H), 2.15
+
5.56 Hz, 1 H), 9.15 (s, 1 H). HRMS (M + H] ) calcd: 673.33442
(s, 3 H), 2.24-2.38 (m, 6 H), 2.38-2.45 (m, 3 H), 3.64 (s, 6 H),
3.74-3.88 (m, 8 H), 6.62 (dd, J ) 8.65, 2.59 Hz, 1 H), 6.73 (d, J
) 2.53 Hz, 1 H), 6.94 (s, 2 H), 7.02-7.17 (m, 5 H), 7.34 (d, J )
8.59 Hz, 1 H), 8.37 (d, J ) 5.68 Hz, 1 H), 9.14 (s, 1 H). MS (ESI,
found: 673.33418. Anal. (C36
,6-Dimethylphenyl 2-((3,5-Bis(methyloxy)-4-((2-(4-morpholi-
nyl)ethyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)-
H
44
N
6
7
O ‚0.7 H
2
O): C, H, N.
2
1
+
phenyl)carbamate (40). H NMR (400 MHz, DMSO-d
.08 (s, 6 H), 2.40-2.48 (m, 4 H), 2.54-2.63 (m, 2 H), 3.51-
.60 (m, 4 H), 3.63 (s, 6 H), 3.78 (s, 3 H), 3.81 (s, 3 H), 3.83-
.90 (m, 2 H), 6.57-6.65 (m, 1 H), 6.72 (d, J ) 2.53 Hz, 1 H),
.94 (s, 2 H), 7.02-7.16 (m, 4 H), 7.34 (d, J ) 8.59 Hz, 1 H),
.36 (d, J ) 5.68 Hz, 1 H), 9.16 (s, 1 H). HRMS (M + H] )
calcd: 660.30279 found: 660.30315. Anal. (C35
O): C, H, N.
,6-Dimethylphenyl-2,4-bis(methyloxy)phenyl(2-((4-((3-(1-pi-
peridinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl)carbamate
6
) δ ppm
pos. ion) m/z: 687 (M + 1). HRMS (M + H] ) calcd: 687.35007
2
3
3
6
8
found: 687.34959. Anal. (C37
H N
46 6
7 2
O ‚0.3 H O): C, H, N.
Lck Kinase Assay. The Lck HTRF kinase assay involved ATP-
dependent phosphorylation of a biotinylated substrate peptide of
gastrin in the presence or absence of inhibitor compound. The final
concentration of gastrin was 1.2 µM. The final concentration of
+
H
41
N
5
O
8
‚0.7
ATP was 0.5 µM (K
m
app ) 0.6 ( 0.1 µM), and the final
H
2
concentration of Lck (a GST-kinase domain fusion (AA 225-509))
was 250 pM. Buffer conditions were as follows: 50 mM HEPES
pH ) 7.5, 50 mM NaCl, 20 mM MgCl, 5 mM MnCl, 2 mM DTT,
0.05% BSA. The assay was quenched and stopped with 160 µL of
2
1
(
6
41). H NMR (400 MHz, DMSO-d ) δ ppm 1.34-1.42 (m, 2 H),