6886 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Cha et al.
(S)-1-Acryloyl-N-[4-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl-
amino)-7-(2-methoxyethoxy) quinazolin-6-yl]pyrrolidine-2-carbox-
amide (19a). Pale yellow solid, 60% yield. 1H NMR (DMSO-d6,
300 MHz): δ 10.61 (s, 1H), 9.71 (s, 1H), 9.05 (s, 1H), 8.59 (d, 1H),
8.45 (s, 1H), 8.31 (s, 1H), 7.93 (d, 1H), 7.88 (td, 1H), 7.64 (dd, 1H),
7.58 (d, 1H), 7.37 (m, 2H), 7.28 (s, 2H), 7.23 (d, 1H), 5.28 (s, 2H),
4.36 (t, 2H), 3.82 (m, 1H), 3.79 (t, 2H), 3.40 (s, 3H), 2.68 (m, 2H),
2.10 (m, 2H), 1.67 (m, 2H).
(S)-1-Acryloyl-N-[4-(3-chloro-4-fluorophenylamino)-7-(2-meth-
oxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide (19b). White
solid, 28% yield. 1H NMR (DMSO-d6, 300 MHz): δ 9.83 (s, 1H),
9.52 (s, 1H), 8.81 (s, 1H), 8.06 (m, 1H), 7.73 (m, 1H), 7.38 (m, 1H),
7.27 (s, 1H), 6.67 (m, 1H), 6.20 (m, 1H), 5.74 (m, 1H), 4.73 (m, 1H),
4.30 (m, 2H), 3.63 (m, 2H), 3.61 (m, 2H), 3.32 (s, 3H), 2.09 (m, 2H),
1.95 (m, 2H).
(S)-1-Acryloyl-N-[4-(3-chloro-2-fluorophenylamino)-7-(2-meth-
oxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide (19c). White
solid, 10% yield. 1H NMR (CDCl3, 300 MHz): δ 9.99 (s, 1H), 9.07
(s, 1H), 8.68 (s, 1H), 8.39 (dd, 1H), 7.53 (s, 1H), 7.14 (d, 2H), 6.55
(d, 1H), 6.53 (s, 1H), 5.82 (dd, 1H), 4.94 (d, 1H), 4.34 (m, 2H), 3.96
(m, 2H), 3.77 (td, 1H), 3.62 (m, 1H), 3.50 (s, 3H), 2.64 (m, 1H),
2.14 (m, 2H), 1.95 (m, 1H); 13C NMR (125 MHz, CDCl3): δ 169.8,
166.1, 156.4, 154.0, 152.7, 148.3, 129.2, 129.1, 127.9, 124.8, 124.4,
122.3, 121.8, 121.0, 109.6, 109.0, 107.5, 70.4, 68.4, 60.9, 59.1, 47.5,
26.9, 25.1; HRMS (FABþ) m/z calcd for C25H25ClFN5O4 [M þ
H]þ: 514.1657. Found: 514.1658.
1.88 mmol) in ethyl acetate (20 mL) was slowly added metha-
nesulfonic acid (0.27 mL, 4.14 mmol). After being stirred at
room temperature for 2 h, the regenerated solid was collected
through simple filtration with quick wash by cooled ethyl
acetate and dried under reduced pressure. Ivory solid, 86%
yield. 1H NMR (CDCl3, 500 MHz): δ 9.85 (s, 1H), 9.09 (s, 1H),
8.53 (s, 1H), 7.61 (m, 1H), 7.56 (s, 1H), 7.07 (m, 1H), 6.53 (dd,
1H), 6.44 (d, 1H), 5.85 (dd, 1H), 4.89 (t, 1H), 4.34 (m, 1H), 4.23
(m, 1H), 3.90 (m, 1H), 3.79 (m, 2H), 3.65 (m, 1H), 3.42 (s, 3H),
2.91 (s, 6H), 2.41 (m, 1H), 2.11 (m, 3H); 13C NMR (125 MHz,
CDCl3): δ 170.2, 166.4, 159.5, 155.7, 154.6, 148.8, 136.8, 130.4,
130.1, 127.4, 125.8, 112.6, 111.8, 111.7, 110.9, 106.7, 100.3, 70.0,
69.8, 61.2, 58.9, 47.9, 39.3, 27.8, 24.9.
General Procedures for the EGFR Enzyme Assay. A total of
10 μL of EGFR enzyme (Her-1, Her-2, or Her-1 T790 M kinase,
Upstate) was added to each well of a 96-well microplate. As an
EGFR inhibitor, 10 μL of serially diluted solution of the
synthesized compounds was added to the individual wells, and
the plate was incubated at room temperature for 10 min. A total
of 10 μL of Poly (Glu, Tyr 4:1, Sigma) and 10 μL of ATP were
successively added to initiate a kinase reaction, and the resulting
mixture was incubated at room temperature for 1 h. A total of
10 μL of 100 mM EDTA was added to each well and stirred for
5 min to terminate the kinase reaction. Then 10 μL of 10ꢀ
antiphosphotyrosine antibody (Pan Vera), 10 μL of 10ꢀ protein
tyrosine kinase (PTK) green tracer (Pan Vera), and 30 μL of
fluorescence polarization (FP)-diluted buffer were added to the
reacted mixture, followed by incubation in the dark at room
temperature for 30 min. The FP value of each well was deter-
mined using a VICTORIII fluorescence meter (Perkin-Elmer) at
488 nm. The IC50, that is, the concentration at which 50%
inhibition was observed, was determined by setting the max-
imum value (0% inhibition) to the polarized light value for a well
untreated with EGFR inhibitor and the minimum value (100%
inhibition). IC50 calculations and analysis were carried out using
Microsoft Excel.
General Procedures for the Cell Growth Inhibitory Assay. A
human vaginal epidermoid cancer cell line, A431 (ATCC: CRL-
1555), a human breast cancer cell line, SK-Br3 (ATCC: HTB-
30), and a primary human fibroblast cell-line, Hs27 (ATCC:
CCL-34), were used to measure the inhibitory activities of the
synthesized compounds toward cancer cell growth and cyto-
toxicity to normal cells. Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with 4.5 g/L glucose and
1.5 g/L sodium bicarbonate, and were supplemented with 10%
fetal bovine serum (FBS). In addition, an Gefitinib/Erlotinib-
resistant non-small cell lung cancer cell line, H1975 (ATCC:
CRL-5908), was incubated in an RPMI medium containing 1%
sodium pyruvate and 10% FBS. These cell lines, stored in a
liquid nitrogen tank, were quickly thawed at 37 °C and centri-
fuged to remove the medium. The resulting cell pellet was mixed
with the culture medium and incubated in a culture flask at 37 °C
under 5% CO2 for 2-3 days; thereafter, the medium was
removed. The remaining cells were washed with Dulbecco’s
phosphate buffered saline (DPBS) and were separated from
the flask using Trypsin-EDTA. The separated cells were diluted
with the culture medium to a concentration of 100000 cells/mL
for A431 or Hs27 or 200000 cells/mL for SK-Br3. A total of 100
μL of the diluted cell solution was added to each well of a 96-well
plate and incubated at 37 °C under 5% CO2 for 1 day.
The synthesized compounds were dissolved in 99.5% DMSO
to a concentration of 25 mM. If the test compound was not
soluble in DMSO, a small amount of 1% HCl was added and the
mixture was held in a 40 °C water bath for 30 min until complete
dissolution was achieved. The test compound solution was
diluted with culture medium to a final concentration of 100
μM and then diluted 10 times serially to 10-6 μM (the final
concentration of DMSO was less than 1%). The medium was
then removed from each of 96 wells of the microplate. A total of
100 μL of the test compound solution was added to each well
(S)-1-Acryloyl-N-[4-(3-chloro-2,4-difluorophenylamino)-7-(2-
methoxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide (19d).
1
White solid, 14% yield. H NMR (CDC13, 300 MHz): δ 9.95
(s, 1H), 9.03 (s, 1H), 8.60 (s, 1H), 8.06 (m, 1H), 7.62 (bs, 1H), 7.21
(s, 1H), 7.01 (m, 1H), 6.53 (m, 2H), 5.82 (m, 1H), 4.94 (m, 1H),
4.30 (m, 2H), 3.94 (m, 2H), 3.76 (m, 1H), 3.62 (m, 1H), 3.49 (s,
3H), 2.58 (m, 1H), 2.33 (m, 1H), 2.13 (m, 2H), 1.95 (m, 1H); 13
C
NMR (125 MHz, CDCl3 þ CD3OD, 1 drop): δ 170.1, 170.0,
166.0, 157.3, 154.2, 152.7, 152.6, 148.1, 129.3, 128.3, 127.8, 123.8,
123.7, 111.2, 111.1, 110.0, 109.3, 107.1, 70.3, 68.2, 60.9, 59.0, 47.5,
27.4, 25.0; HRMS (FABþ) m/z calcd for C25H24ClF2N5O4 [M þ
H]þ: 532.1563. Found: 532.1575.
(S)-1-Acryloyl-N-[4-(3,4-dichloro-2-fluorophenylamino)-7-(2-
methoxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide (19e).
1
White solid, 10% yield. H NMR (CDC13, 300 MHz): δ 10.03
(s, 1H), 9.06 (s, 1H), 8.67 (s, 1H), 8.36 (t, 1H), 7.52 (s, 1H), 7.30
(dd, 1H), 7.24 (s, 1H), 6.55 (m, 2H), 5.83 (m, 1H), 4.95 (m, 1H),
4.33 (m, 2H), 3.95 (m, 2H), 3.76 (m, 1H), 3.62 (m, 1H), 3.51 (s,
3H), 2.61 (m, 1H), 2.13 (m, 2H), 1.97 (m, 1H).
(S)-1-Acryloyl-N-[4-(4-bromo-3-chloro-2-fluorophenylamino)-
7-(2-methoxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide
1
(19f). Ivory solid, 31% yield. H NMR (CDCl3, 300 MHz): δ
9.99 (s, 1H), 9.05 (s, 1H), 8.65 (s, 1H), 8.20 (t, 1H), 7.70 (bs, 1H),
7.43 (dd, 1H), 7.25 (s, 1H), 6.55 (m, 2H), 5.83 (m, 1H), 4.94 (m,
1H), 4.32 (m, 2H), 3.94 (m, 2H), 3.78 (m, 1H), 3.60 (m, 1H), 3.5
(s, 3H), 2.61 (m,1H), 2.10 (m, 3H).
(S)-1-Acryloyl-N-[4-(3-chloro-2-fluorophenylamino)-7-(2-meth-
oxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide Hydrochlor-
ide (19c HCl). To a stirred solution of 19c (1.0 g, 1.95 mmol) in
isopropanol (20 mL) was slowly added 1 N HCl ethereal solution
(4.88 mL, 4.88 mmol). After being stirred at room temperature for
1 h, the regenerated solid was collected through simple filtration
with quick wash by cooled isopropanol and dried under reduced
pressure. White solid, 94% yield. 1H NMR (CDCl3, 300 MHz): δ
10.38 (s, 1H), 9.22 (s, 1H), 8.80 (s, 1H), 8.66 (s, 1H), 8.04 (s, 1H),
7.90 (t, 1H), 7.39 (t, 1H), 7.25 (td, 1H), 6.54 (d, 2H), 5.85 (t, 1H),
4.95 (d, 1H), 4.45 (m, 2H), 3.95 (m, 2H), 3.70 (m, 1H), 3.50 (m,
1H), 3.48 (s, 3H), 2.50 (m, 1H), 2.14 (m, 2H), 2.00 (m, 1H); 13
C
NMR (125 MHz, CDCl3): δ 170.4, 166.0, 159.3, 155.0, 153.5,
151.5, 148.4, 136.8, 130.3, 129.5, 129.3, 127.9, 126.0, 124.6, 121.9,
111.6, 106.9, 100.2, 70.0, 69.6, 61.0, 59.1, 47.9, 27.4, 25.2.
(S)-1-Acryloyl-N-[4-(3-chloro-2,4-difluorophenylamino)-7-(2-
methoxyethoxy)quinazolin-6-yl]pyrrolidine-2-carboxamide Me-
sylate (19d Mesylate). To a stirred solution of 19d (1.0 g,