Discovery of novel c-Met kinase inhibitors bearing a thieno[2,3-d] pyrimidine or furo[2,3-d]pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmc.2011.05.038

A series of thieno[2,3-d]pyrimidines and furo[2,3-d]pyrimidines were synthesized and evaluated for the c-Met inhibition. Thieno[2,3-d]pyrimidine 6b stood out as the most potent showing an IC₅₀ of 35.7 nM. This compound displayed high inhibitory effect on cell proliferation in BaF3-TPR-Met cells and showed high selectivity for c-Met family against other 14 tested kinases. However, compound 6b was found ineffective in the c-Met-dependent U-87MG human gliobastoma xenograft model that may be relevant to its poor PK profile.

Full text of DOI:10.1016/j.bmc.2011.05.038

Bioorganic & Medicinal Chemistry 19 (2011) 3906–3918
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel c-Met kinase inhibitors bearing a thieno[2,3-d]pyrimidine
or furo[2,3-d]pyrimidine scaffold
Ailing Zhao a, , Xin Gao b, , Yuanxiang Wang a, Jing Ai b, Ying Wang b, Yi Chen b, Meiyu Geng b, , Ao Zhang a,
⇑
⇑
a Synthetic Organic and Medicinal Chemistry Laboratory (SOMCL), State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences,
Shanghai 201203, China
b Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thieno[2,3-d]pyrimidines and furo[2,3-d]pyrimidines were synthesized and evaluated for the
c-Met inhibition. Thieno[2,3-d]pyrimidine 6b stood out as the most potent showing an IC50 of 35.7 nM.
This compound displayed high inhibitory effect on cell proliferation in BaF3-TPR-Met cells and showed
high selectivity for c-Met family against other 14 tested kinases. However, compound 6b was found inef-
fective in the c-Met-dependent U-87MG human gliobastoma xenograft model that may be relevant to its
poor PK profile.
Received 6 April 2011
Revised 19 May 2011
Accepted 19 May 2011
Available online 24 May 2011
Keywords:
RTK
Ó 2011 Elsevier Ltd. All rights reserved.
c-Met
Thieno[2,3-d]pyrimidine
Furo[2,3-d]pyrimidine
Antitumor
1. Introduction
(IC50, 27 nM) and favorable pharmacokinetic characteristics. It
exhibited remarkable tumor regression at an oral dosage of
c-Met is a member of the receptor tyrosine kinase (RTK) family.
It is the cell surface receptor for hepatocyte growth factor (HGF),
and is normally expressed by epithelial cells of many organs during
embryogenesis and in adulthood.1,2 Aberrant HGF/c-Met signaling
has been found affecting cell proliferation, survival and motility,
leading to tumor growth, angiogenesis and metastasis.3–9 There-
fore, c-Met has emerged as a promising therapeutic target for can-
cer therapy. Many small molecules selectively targeting the ATP
binding site of c-Met kinase have been identified and exerted
significant therapeutic effects in treating human cancers
clinically.10–15 Among these, acylthiourea 115 developed by Kirin
(Fig. 1) represented one of the earliest c-Met inhibitors possessing
potent inhibition of c-Met phosphorylation in epidermoid carci-
noma cells with IC50 value of 121 nM, and showing inhibition to tu-
mor growth by 70% at a dosage of 100 mg/kg against human brain
tumor cell (U-87MG) transplanted in nude mice.15 As a structural
mimic of the widely used quinoline scaffold in 1, MethylGene
Inc.16,17 recently reported a series of c-Met inhibitors by replacing
the quinoline core with a thieno[3,2-b]pyridine framework. The
lead compound 2a showed significantly enhanced in vitro activity
40 mg/kg in the MKN-45 human xenograft mouse model.17 Mean-
while, Bristol-Myers Squibb18 reported a series of pyrrolopyridines,
also as analogues of 1, among which compound 2b displayed
highly potent c-Met potency (IC50, 1.8 nM) and demonstrated sig-
nificant in vivo antitumor activity in the GTL-16 human gastric car-
cinoma xenograft model at doses of 25–100 mg/kg. Interestingly,
all compounds 1, 2a, and b showed significant potency as well
for the vascular endothelial growth factor receptor (VEGFR), an-
other key factor implicated in the development and progression
of various human cancers. Therefore, to clarify the therapeutic ben-
efit of c-Met-targeted drugs, potent and selective c-Met inhibitors
are highly needed.
During drug screening of our internal compound libraries, we
found that thieno- or furo-[2,3-d]pyrimidine cores (I or II, Fig. 1)
that were widely used in other categories of therapies displayed
potent and selective c-Met inhibitory activity upon introducing
an appropriate aryloxy substituent at the C4- as that in 2b. Herein,
we report the synthesis and pharmacological evaluation of these
two novel series of compounds.
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel./fax: +86 21 50806072 (M.G.); tel./fax: +86 21
50806035 (A.Z.).
E-mail addresses: mygeng@mail.shcnc.ac.cn (M. Geng), aozhang@mail.shcnc.
ac.cn (A. Zhang).
As described in Scheme 1, the 4,5-disubstituted thieno
[2,3-d]pyrimidine series was prepared from the key intermediates
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.038

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3907
H
N
H
N
H
R1
N
R3
N
R2
H
N
R3
S
O
O
O
N
O
O
O
N
O
R
4
O
O
F
5
F
Y
F
N
S
F
N
N
X
N
2
N1
I (X=S)
II (X=O)
Et
H
1 (Kirin)
2a (MethylGene)
c-Met:IC50 =27nM
VEGFR-2:IC50 =7nM
2b (BMS)
c-Met:IC50 =121nM
c-Met:IC50 =1.8nM
VEGFR-2:IC50 =27nM
VEGFR-2:IC50 =32nM
Our proposed new scaf fold
(R=H,Alkyl,Ar)
H
(Y=Alkyl,alkoxy,oramino)
N
N
N
N
R1 =:
R2 =:
R3 =:
;
;
O
O
O
F
F
Figure 1. Representative c-Met inhibitors (1 and 2) and our proposed thieno- and furo-[2,3-d]pyrimidines I and II.
F
F
H
N
N
NH2
R
O
N
Cl
N
O
O
R
R
O
O
R
F
i
NH
Cl
ii
N
iii
N
N
S
S
6a-j
S
S
N
N
3a-j
4a-j
5a-j
a (R = H, 50%); c (R = 4-MeO-Ph, 65%);
b (R = Ph, 53%); d (R = 5-Me-thiophen-2-yl, 81%);
e (R = 3,4-diMeO-Ph, 64%); f (R = 4-biphenyl, 58%);
R'
g (R = 2-naphtnyl, 54%); h (R = 4-CF3-Ph, 59%);
ii,iii
ii,iii
N
N
j (R= 3,4-diF-Ph, 55%)
i (R =4-F-Ph, 46%);
Ph
Ph
S
S
8
N
N
7
(63%)
F
H
N
N
Cl
R'
R' =
O
O
O
n
n
N
N
F
S
S
N
X
N
X
9a
9b
(n = 1, X = H)
(n = 2, X = Me)
10a
10b (57%)
10c (62%)
(53%)
9c (n = 3, X = H)
Scheme 1. Reagents and conditions: (i) POCl3, 100 °C, 1 h, ꢀ50%; (ii) 4-amino-2-fluorophenol, NaH, DMF, 0 °C, 2 h, ꢀ90%; (iii)1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-
3-carboxylic acid, TBTU, DIPEA, DMF, 0 °C, 4 h.
O
N
Cl
N
OH
R
R
R
H
iii, iv
NH
N
N
i
ii
Cl
+
NO2
11
O
O
HO
N
12a-c
X
X
X
13a-f
14a-f (50~65%)
(X =: a, H; b, Me; c, SMe)
F
H
N
O
N
O
N
Ph
O
Ph
From 15e
O
O
N
vi-viii
O
R
N
F
v
N
O
S
N
O
X
O
O
X
N
16 (79%)
17
18
19
(X = NHMe, 25%)
(X = N3, 30%)
(X = MeO, 44%)
15a (R = Me, X = H, 61%); 15b (R = Ph, X = H, 68%);
15c (R = thiophen-2-yl, X = H, 40%);
15d (R = thiophen-2-yl, X = Me, 67%);
15e (R = Ph, X = MeS, 44%); 15f (R = 4-F-Ph, X = H, 49%)
Scheme 2. Reagents and conditions: (i) DBU, EtOH, 120 °C, overnight, 20–50%; (ii) POCl3, 100 °C, 0.5 h, ꢀ50%; (iii) 4-amino-2-fluorophenol, NaH, DMF, 0 °C, 0.5 h, 60–90%; (iv)
1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid, TBTU, DIPEA, DMF, 0 °C to rt, 4 h, 40–70%; (v) mCPBA, DCM, 0 °C, 2 h; (vi) CH3NH2ꢁHCl, DIPEA, DMF, 140 °C,
microwave, 1 h; (vii) NaN3, DMF, rt, 12 h; (viii) NH3ꢁCH3OH, rt, overnight.

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A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
Table 1
c-Met enzymatic activity of selected compoundsa
Compound
Structure
c-Met inhibition
% at 10
lM
IC50 (nM)
O
N
F
F
NH
O
6a
14.3
>10 lM
O
N
S
N
O
N
F
F
NH
O
6b
6c
6d
6e
6f
94.3
90.0
85.5
99.9
80.4
92.1
83.8
35.7 3.6
46.5 6.2
42.0 10.4
123 77.1
O
N
S
N
O
N
F
H3CO
F
NH
O
O
N
N
O
S
N
F
F
NH
O
O
S
N
S
N
O
N
F
H3CO
H3CO
F
NH
O
O
N
S
N
O
N
F
F
NH
O
>1 l
Mb
O
N
O
S
N
N
F
F
NH
O
6g
>1 l
Mb
O
N
S
N
O
N
F
F3C
F
NH
O
6h
190.8 40.7
O
N
S
N

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3909
Table 1 (continued)
Compound
Structure
c-Met inhibition
% at 10
l
M
IC50 (nM)
O
N
F
F
F
NH
O
6i
71.9
68.0 11.1
O
N
S
N
O
N
F
F
F
F
NH
O
6j
88.4
10.6
12.3
31.2
23.4
65.7 1.4
O
N
S
N
O
N
F
F
NH
O
8
>10
>10
>10
>10
>10
lM
lM
lM
lM
lM
O
N
N
S
O
N
F
F
NH
O
10a
10b
10c
O
N
S
N
O
N
F
F
NH
O
O
N
N
S
O
N
F
F
NH
O
O
N
N
S
O
N
F
F
NH
O
15a
16.5
O
N
O
N
O
N
F
F
NH
O
15b
84.9
69.8 11.6
O
N
N
O
(continued on next page)

3910
A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
Table 1 (continued)
Compound
Structure
c-Met inhibition
% at 10
lM
IC50 (nM)
O
N
F
F
NH
O
15c
15d
15e
15f
46.6
>10
>10
>10
l
l
l
M
M
M
O
S
N
O
N
O
N
F
F
NH
O
24.3
33.4
80.9
O
S
N
O
N
O
N
F
F
NH
O
O
N
N
O
S
O
N
F
F
F
NH
O
310 138
O
N
N
O
O
N
F
F
NH
O
16
36.7
>10 lM
O
N
N
O
S
O
O
O
N
F
F
NH
O
17
78.2
>1
l
Mb
O
N
O
N
N
H
O
N
F
F
NH
O
18
18.5
>10 lM
O
N
O
N
N3
O
N
F
F
NH
O
19
31.7
>10 lM
O
N
O
N
O
1
2b
—
—
—
—
12116
1.820
a
IC50s were calculated by Logit method from the results of at least three independent tests with six concentrations each and expressed as means SD.
Inhibitory effect less than 50% at 1 lM concentration.
b

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3911
4a–j, which were either obtained from commercial source or
prepared by chlorination19 of thieno[2,3-d]pyrimidin-4(3H)-one
(3a–j) in refluxed POCl3 in moderate yields. Condensation20 of
chlorides 4a–j with 4-amino-2-fluorophenol afforded diaryl ethers
5a–j in 90% yield. Treating18 amines 5a–j with 1-(4-fluorophenyl)-
2-oxo-1,2-dihydropyridine-3-carboxylic acid under TBTU and
DIPEA yielded the target compounds 6a–j in 46–81% yields.
4,6-Disubstituted thieno[2,3-d]pyrimidine 8 was prepared from
chloride 7 in 63% yield. Similarly, thieno[2,3-d]pyrimidines 10a–c
bearing a 5,6-cyclic substituent or a 2-methyl group were prepared
from corresponding chlorides 9a–c in a similar manner in 53–62%
yields.
Since chlorides 14a–f (Scheme 2), the key intermediates to
access furo[2,3-d]pyrimidines 15a–f were not commercially avail-
able, they were prepared21 from 2-amino-4-methylfuran-3-carbo-
nitrile 11 and 4,6-dihydroxypyrimidines 12a–c leading to furo[2,
3-d]pyrimidinones 13a–f in moderate yields. Treating18 13a–f with
refluxed POCl3 provided the chlorides 14a–f in 50–65% yields. Sim-
ilarly, treating19 chlorides 14a–f with 4-amino-2-fluorophenol
following by condensation20 with 1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxylic acid under TBTU and DIPEA yielded
target compounds 15a–f in 33–83% overall yields. Further, oxida-
tion22 of 15e with mCPBA provided sulfone 16 in 79% yield. Treat-
ing sulfone 16 with methylamine under microwave irridation23
provided 17 in 25% yield. Reaction24 of sulfone 16 with NaN3 in
DMF was very sluggish and yielded azide 18 in 30% yield. React-
ing25 sulfone 16 with ammonia in MeOH did not yield the corre-
sponding 2-amino product, instead 2-methoxy substituted
furo[2,3-d]pyrimidine 19 was obtained in 44% isolated yield.
thieno[2,3-d]pyrimidine 6b. It is quite disappointing that replacing
the C5-phenyl with a thien-2-yl (15c, d) or introducing a substitu-
ent at the C2 position (15e, 16–19) completely abolished the c-Met
activity. 5-(4-Fluorophenyl)-furo[2,3-d]pyrimidine 15f retained
moderate c-Met potency with an IC50 value of 310 nM. In
comparison of the potency between 6b and 15b, 6i and 15f, thie-
no[2,3-d]pyrimidines 6b and i are generally more potent than their
furo[2,3-d]pyrimidine congeners 15a and f.
From the results above, thieno[2,3-d]pyrimidines 6b–d, i, j and
furo[2,3-d]pyrimidine 15b stood out as the potent c-Met inhibitors
with IC50 ranging between 35–70 nM. These compounds were
more potent than the Kirin’s initial compound 1, but less potent
than Methylgene’s compound 2a and BMS compound 2b (Table 1).
2.3. Cell proliferation study
Since activation of c-Met ultimately results in cell proliferation,
we then evaluated the inhibitory effect of the potent compounds
6a–d, i, j, and 15b on cell proliferation at concentrations of 10, 1,
and 0.1 lM in TPR-Met transformed BaF3 cells (BaF3-TPR-Met)
that stably express a constitutively active, ligand-independent,
oncogenic form of c-Met.26,31 As shown in Table 2, treatment of
BaF3-TPR-Met cells with thieno[2,3-d]pyrimidines 6b–d, i, and j
at 10
compounds 6b–d retained good inhibitory effects at 1
tration. In agreement with the enzymatic results, all the thie-
no[2,3-d]pyrimidines with C5-aryl substituent had higher
lM significantly inhibited cell growth (>80%), while only
lM concen-
a
inhibition capacity than the C5-nonsubstituted analog 6a that is
inactive at all tested concentrations. Furo[2,3-d]pyrimidine 15b
showed good inhibitory effect at 10 lM concentration, however
such effect was completely lost at the concentration of 1 lM.
2.2. c-Met enzymatic activity
2.4. c-Met phosphorylation study
All the newly synthesized thieno[2,3-d]pyrimidines 6a–j, 8,
10a–c, and furo[2,3-d]pyrimidines 15a–f, 16–19 were evaluated
for their ability to inhibit enzymatic activity of the c-Met receptor
using a procedure similar to that we reported26 recently or as that
in the literature.27–30 The results are summarized in Table 1.
Among the thieno[2,3-d]pyrimidine series, C5-nonsubstituted ana-
logue 6a is inactive for c-Met, while wide differences in potency
were observed for the C5-aryl substituted compounds 6a–j. 5-Phe-
nyl-, 5-(4-methoxyphenyl)-, and 5-(5-methyl-thien-2-yl)-substi-
tuted analogs 6b–d displayed high c-Met inhibitory potency with
similar IC50 values of 35.7, 46.5, 42.0 nM, respectively. These
results indicated that the C5-aryl substituent in the thieno[2,3-
d]pyrimidine scaffold may be fitting into a lipophilic binding
pocket in c-Met. 3,5-Dimethoxyphenyl substituted congener 6e
displayed slightly (3-fold) decreased potency with an IC50 value
of 123 nM, suggesting that a bulky C5-substituent is not well toler-
ated in the proposed lipophilic pocket. This was further supported
by the significant reduction in c-Met potency of compounds 6f and
g that contain a bulkier substituent. Good potency was retained on
4-fluorophenyl- and 3,4-difluorophenyl substituted thieno[2,3-
d]pyrimidines 6i and j, which showed IC50 values of 68.0 and
65.7 nM, respectively. This result indicated that the electronic
property of the substituent in the C5-aryl does not have significant
effect on the c-Met potency. It is noteworthy that the C6-aryl
substituted thieno[2,3-d]pyrimidine 8 is nearly inactive at the
c-Met. Similarly, thieno[2,3-d]pyrimidines 10a–c bearing a 5,6-
cyclic substituent were also inactive. These results indicated that
a substituent at the C6 in the thieno[2,3-d]pyrimidine core is not
tolerated for c-Met.
From the results obtained above, thieno[2,3-d]pyrimidines
6b–d were confirmed active in both c-Met enzymatic and cell
growth assays. To determine whether c-Met kinase inhibition of
these compounds in cell-free system could be recapitulated in vi-
tro, the inhibition of tyrosine phosphorylation of cellular proteins
Table 2
Effect of the newly synthetic compounds on cell proliferation
Compounds
Inhibition rate (%)
10
lM
1
lM
0.1 lM
6a
5.4
5.3
ꢂ11.8
ꢂ6.5
76.5
69.5
91.1
92.6
60.6
64.5
30.8
33.3
13.9
33.7
14.6
14.7
ꢂ9.3
ꢂ3.0
ꢂ2.8
6.0
ꢂ4.7
ꢂ12.8
ꢂ3.4
ꢂ10.5
ꢂ10.1
4.1
ꢂ2.3
ꢂ4.1
12.9
13.7
ꢂ1.1
4.6
ꢂ4.1
ꢂ6.9
ꢂ2.7
ꢂ12.1
0.6
29.5
10.3
11.7
ꢂ4.7
2.8
6b
94.7
94.6
94.3
94.5
95.3
95.0
92.2
94.5
92.3
89.7
93.7
94.4
ꢂ5.4
ꢂ4.7
6.2
6c
6d
6i
6j
15b
15c
15d
15e
15f
ꢂ9.7
14.9
15.1
77.7
76.3
5.8
2.0
6.1
ꢂ5.8
Among the furo[2,3-d]pyrimidine subseries, C5-methyl substi-
tuted analogue 15a is inactive at the c-Met, whereas C5-phenyl
substituted congener 15b displayed high c-Met inhibitory potency
with IC50 value of 69.8 nM, only 2-fold less potent than that of
The inhibition ratio (%) of cell proliferation was calculated using the following
equation: ½1 ꢂ ðA570=A570 controlÞꢃ ꢄ 100%.26

3912
A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
Figure 2. Dose-dependent inhibition of the selected compounds on c-Met phosphorylation in BaF3-TPR-Met cells.
by these compounds in TPR-Met-transformed BaF3 cells were
investigated, furo[2,3-d]pyrimidines 15b and f were also evaluated
as comparison. As shown in Figure 2, compounds 6b–d, 15b, and f
inhibited c-Met autophosphorylation in a dose-dependent manner
in BaF3-TPR-Met cells. In general, compound 6b had higher inhib-
itory effects than other compounds in the tested cell lines, there-
fore it was selected for further evaluation.
2.5. Kinase selectivity of lead compound 6b
To examine whether lead compound 6b is a selective c-Met
inhibitor, it was screened against c-Met family member, Ron, along
with other 14 tyrosine kinases, including RON, Flt-1, c-Kit, PDGFRa,
PDGFRb, RET, EGFR, ErbB2, ErbB4, c-Src, Abl, EPH-A2, EPH-B2,
IGF1R, and FGFR1. Compared to its high potency against c-Met
(IC50, 35.7 nM) and RON (IC50, 50.4 nM), compound 6b produced
more than 10,000-fold less potency against other selected kinases,
Figure 3. Dose-dependent inhibition of compound 6b on c-Met phosphorylation
and signal transduction pathways in BaF3-TPR-Met cells.
with IC50 values greater than 10 lM (Table 3), indicating that com-
pound 6b is a potent selective c-Met family-targeting inhibitor.
2.6. c-Met downstream signaling study on lead 6b
As shown in Figure 3, exposure to compound 6b produced sig-
nificant inhibition of c-Met phosphorylation in BaF3-TPR-Met cells.
In addition, the phosphorylation of Erk1/2 and AKT, the key mole-
cules downstream of c-Met that play important roles in cellular
proliferation,2,28 also were significantly inhibited upon treatment
with compound 6b. These data further supported that compound
6b inhibits c-Met activity as well as subsequent c-Met downstream
signaling.
2.7. Antitumor study on lead 6b
To evaluate the inhibition of compound 6b on c-Met activities
in vivo, the antitumor activity of compound 6b was investigated
in the c-Met-dependent U-87MG human gliobastoma xenograft
model. We found that compound 6b, even at the dosage of
100 mg/kg, only showed a marginal effect on tumor growth
(Fig. 4). Compound 6b was well tolerated showing no significant
loss of body weight in this xenograft models (data not shown).
Figure 4. Antitumor efficacy of compound 6b in the U-87MG xenograft models.
Tumor-bearing nude mice were randomly divided in groups when tumor volume
reached 100–200 mm3 and given compound 6b ip at indicated dose levels or
vehicle alone over the designated treatment schedule. Data are presented as
means SEM. n = 6 mice per group.
Compound 6b displayed a short half-life time (1.67 h), moderate
clearance (2.83 L/h/kg, iv), and poor oral bioavailability (5%). This
unfavorable PK profile of thieno[2,3-d]pyrimidines 6b along with
its poor aqueous solubility may be the major reason for the poor
antitumor efficacy. Therefore, more focused structural optimiza-
tion on the solubility is needed.
2.8. Pharmacokinnetic (PK) profile of lead 6b
The PK parameters of compound 6b were then evaluated in
mice after single iv (10 mg/kg) and po (20 mg/kg) administration.
Table 3
3. Conclusion
Kinase selectivity of compound 6b
Kinase
Enzyme IC50 (nM)
Kinase
Enzyme IC50 (nM)
In summary, a series of thieno[2,3-d]pyrimidines and furo[2,3-
d]pyrimidines were synthesized and evaluated for the c-Met inhi-
bition. Thieno[2,3-d]pyrimidine 6b stood out as the most potent
c-Met inhibitor with an IC50 of 35.7 nM. This compound displayed
high inhibitory effect on cell proliferation in BaF3-TPR-Met cells
and showed high selectivity for c-Met family against other 14
tested kinases. However, compound 6b was found ineffective in
the c-Met-dependent U-87MG human gliobastoma xenograft mod-
RON
c-Kit
PDGFR
PDGFRb
RET
EGFR
ErbB2
ErbB4
50.4 1.6
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
c-Src
ABL
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
a
EPH-A2
EPH-B2
IGF1R
FGFR1
Flt-1

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3913
el. The poor PK profile of 6b is likely a critical factor to the ineffec-
tive antitumor efficacy and will be a major objective for further
structural optimization. Meanwhile, further evaluation on other
mouse models such as MKN-45 (effective for 2a) and GTL-16
(effective for 2b) xenograft models will be conducted as well.
(300 MHz, CDCl3 + CD3OD) d: 7.02 (t, J = 9.0 Hz, 2H), 7.09 (s, 1H),
7.45 (t, J = 9.0 Hz, 2H), 7.86 (s, 1H).
4.1.1.4. Preparation of 4-chloro-5-(4-fluorophenyl)thieno[2,3-
d]pyrimidine (4i).
A solution of 5-(4-fluorophenyl)thie-
no[2,3-d]pyrimidin-4(3H)-one 3i (320 mg, 1.30 mmol) in POCl3
(8 mL) was heated at 100 °C for 1 h. The mixture was evaporated
under reduced pressure and the residue was added to ice water,
and then extracted with Et2O. The organic layer was separated,
washed with brine, dried over anhydrous Na2SO4, and evaporated
under reduced pressure to give the crude product, which was puri-
fied by chromatography on silica gel to afford product 4i (82 mg,
23.9%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) d: 7.14
(m, 2H), 7.37 (m, 2H), 7.46 (s, 1H), 8.87 (s, 1H).
4. Experimental
Reactions were performed under a nitrogen atmosphere in dry
glassware with magnetic stirring. The solvents were purified and
dried according to the standard methods prior to use. Commer-
cially available reagents were used without further purification.
1H and 13C NMR spectra were recorded on a Brucker AC300 spec-
trometer using tetramethylsilane as an internal reference. Chemi-
cal shifts are expressed in ppm and J values are given in Hz.
Analytical thin-layer chromatography (TLC) was carried out on
0.2 mm Kieselgel 60F 254 silica gel plastic sheets (EM Science,
Newark). Column chromatography was used for the routine purifi-
cation of reaction products. The column output was monitored
with TLC. Due to the limited solubility, 13C NMR analysis of com-
pounds 6f, g, 17–19 were not applicable. HPLC analysis of these
compounds was conducted on an Agilent 1100 series LC system
(Agilent ChemStation Rev.A.10.02; ZORBAX Eclipse XDB-C8,
4.1.1.5. Preparation of
no[2,3-d]pyrimidin-4-yloxy)aniline (5i).
3-fluoro-4-(5-(4-fluorophenyl)thie-
To the solution of
4-amino-2-fluorophenol (41 mg, 0.32 mmol) in dried DMF (3 mL),
was added NaH (13 mg, 0.54 mmol). The mixture was stirred at
0 °C for 10 min, and then a solution of 4-chloro-5-(4-fluoro-
phenyl)thieno[2,3-d]pyrimidine 4i (50 mg, 0.19 mmol) in dried
DMF (1 mL) was added. The mixture was stirred at 0 °C for 1.5 h.
Ice water (5 mL) was added to quench the reaction and the mixture
was extracted by Et2O (3 ꢄ 10 mL). The organic layer was washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure to obtain the crude product, which was purified by silica
gel column chromatograph to give compound 5i (60 mg, 88.9%) as
a pale yellow solid. 1H NMR (300 MHz, CDCl3) d: 3.76 (s, 2H), 6.43
(d, J = 9.0 Hz, 1H), 6.49 (m, 1H), 6.89 (t, J = 8.4 Hz, 1H), 7.09 (t,
J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.57 (t, J = 8.4 Hz, 2H), 8.63 (s, 1H);
13C NMR (100 MHz, CDCl3) d: 103.2 (d, J = 21.4 Hz), 110.4, 114.8
(d, J = 21.9 Hz), 116.7, 123.0, 124.0, 130.4 (d, J = 13.2 Hz), 131.0
(d, J = 8.2 Hz), 131.6, 134.7, 145.8 (d, J = 9.5 Hz), 153.1, 154.5 (d,
J = 245.1 Hz), 162.5 (d, J = 245.5 Hz), 163.6, 170.1; MS (EI) m/z:
355 (M+).
4.8 mm ꢄ 150 mm, 5
lM, 1.0 mL/min, uv 254 nM, rt) with two sol-
vent systems (MeCN/H2O/TFA, and MeOH/H2O/TFA). All the as-
sayed compounds displayed a purity of 95–99% in both solvent
systems.
4.1. General procedure for preparation of compounds 6a–j
4.1.1. N-(3-fluoro-4-(5-(4-fluorophenyl)thieno[2,3-d]pyrimidin-
4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydro-
pyridine-3-carboxamide (6i)
4.1.1.1. Preparation of 2-(1-(4-fluorophenyl)ethylidene)malon-
onitrile.
To a solution of 4-fluoroacetophenone (1.00 g,
7.24 mmol), malononitrile (0.957 g, 14.48 mmol) and ammonium
acetate (0.446 g, 5.79 mmol) in 20 mL m-xylene, was added 2 mL
of glacial acetic acid. The mixture was heated to reflux for 12 h.
The solvent was evaporated under reduced pressure. The residue
was poured into water and extracted with EtOAc. The organic layer
was separated, washed with brine, dried over Na2SO4, and
evaporated. The crude product was purified by silica gel column
chromatograph to afford 2-(1-(4-fluorophenyl)ethylidene)malono-
nitrile (1.07 g, 79.4%) as a white solid. 1H NMR (300 MHz, CDCl3) d:
2.62 (s, 3H), 7.21 (m, 2H), 7.60 (m, 2H).
4.1.1.6. Preparation of N -(3-fluoro-4-(5-(4-fluorophenyl)thie-
no[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-
1,2-dihydropyridine-3-carboxamide (6i).
To a solution of
aniline 5i (70 mg, 0.20 mmol), 1-(4-fluorophenyl)-2-oxo-1,2-dihy-
dropyridine-3-carboxylic acid (51.3 mg, 0.22 mmol) and 2-(1H-
benzotriazole-1-yl)-1,1,3-tetramethyluroniumtetrafluoroborate
(TBTU) (87.3 mg, 0.26 mmol) in DMF (4 mL) at 0 °C, was added
diisopropylethylamine (93.0 mg, 125 lL, 0.72 mmol). The reaction
was allowed to warm to rt and stirred for 2 h. The mixture was
quenched with ice water (5 mL) and extracted with Et2O
(3 ꢄ 10 mL). The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo to afford the crude product,
which was purified by silica gel column chromatograph to give
the final product 6i (51 mg, 45.5%) as a white solid. 1H NMR
(300 MHz, CDCl3) d: 6.60 (t, J = 7.2 Hz, 1H), 7.07 (m, 3H), 7.27 (m,
3H), 7.35 (s, 1H), 7.40 (m, 2H), 7.58 (m, 3H), 7.93 (d, J = 13.8 Hz,
1H), 8.60 (s, 1H), 8.72 (d, J = 8.1 Hz, 1H), 11.95 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 107.2, 108.3 (d, J = 23.7 Hz), 115.0 (d,
J = 21.4 Hz), 116.2 (d, J = 21.8 Hz), 120.2, 124.5, 125.2, 129.4 (d,
J = 9.1 Hz), 131.5 (d, J = 8.2 Hz), 131.6, 133.6, 134.4 (d,
J = 12.8 Hz), 136.3, 137.2 (d, J = 9.5 Hz), 144.2, 145.1, 152.2, 152.9,
153.0, 153.5 (d, J = 243.2 Hz), 161.7, 161.9, 162.0 (d, J = 245.1 Hz),
162.6, 169.9; MS-EI m/z: 570 (M+); Anal. Calcd for C30H17F3N4O3S:
C, 61.69; H, 3.19; N, 9.59. Found: C, 61.90; H, 3.11; N, 9.49.
4.1.1.2. Preparation of 2-amino-4-(4-fluorophenyl)thiophene-3-
carbonitrile.
To a solution of 2-(1-(4-fluorophenyl)ethyli-
dene)malononitrile (410 mg, 2.20 mmol) and S8 (113 mg,
3.53 mmol) in tetrahydrofuran (40 mL), was added dropwise a
solution of NaHCO3 (241 mg, 2.87 mmol) in H2O (20 mL). The mix-
ture was stirred at 35 °C overnight and then evaporated. The resi-
due was poured into water and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give the crude product,
which was purified by silica gel column chromatograph to afford
2-amino-4-(4-fluorophenyl) thiophene-3-carbonitrile (330 mg,
68.8%) as a white solid. 1H NMR (300 MHz, CDCl3) d: 4.97 (s, 2H),
6.32 (s, 1H), 7.12 (m, 2H), 7.56 (m, 2H).
4.1.1.3. Preparation of 5-(4-fluorophenyl)thieno[2,3-d]pyrimi-
din-4(3H)-one (3i).
A
mixture of 2-amino-4-(4-fluoro-
phenyl)thiophene-3-carbonitrile (210 mg, 0.96 mmol), formic
acid (8 mL) and acetic anhydride (8 mL) was heated at 110 °C for
36 h. The solvent was removed under reduced pressure and the
residue was purified by chromatography on silica gel to afford
the title compound 3i (171 mg, 81.8%) as a white solid. 1H NMR
4.1.2. N-(3-fluoro-4-(thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-
(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide
(6a)
This compound was prepared as a white solid from 5a following
a procedure similar to that of preparation of 6i in 50% yield. 1H

3914
A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
NMR (300 MHz, C5D5N) d: 6.55 (m, 1H), 7.36 (m, 2H), 7.62 (m, 6H),
7.93 (d, J = 6.3 Hz, 1H), 8.49 (d, J = 12.6 Hz, 1H), 8.81 (m, 2H), 12.53
(s, 1H); 13C NMR (100 MHz, C5D5N) d: 107.1, 109.0 (d, J = 23.6 Hz),
116.4, 116.5 (d, J = 23.2 Hz), 118.7, 119.0, 121.7, 123.0, 124.8,
127.2, 129.4 (d, J = 9.2 Hz), 136.8, 138.5 (d, J = 10.2 Hz), 143.2,
145.2, 153.4, 154.9 (d, J = 244.3 Hz), 162.1, 162.6, 162.8 (d,
J = 246.2 Hz), 163.2, 170.2; MS (EI) m/z: 476 (M+); Anal. Calcd for
(m, 2H), 7.28 (m, 3H), 7.34 (s, 1H), 7.39 (m, 2H), 7.61 (dd,
J = 2.4 Hz, J = 6.6 Hz, 1H), 7.95 (dd, J = 2.4, 12.6 Hz, 1H), 8.59 (s,
1H), 8.71 (dd, J = 2.4, 7.2 Hz, 1H), 11.96 (s, 1H). 13C NMR
(400 MHz, CDCl3) d: 55.7, 55.8, 107.2, 109.2 (d, J = 23.2 Hz),
110.5, 112.9, 116.0, 116.7, 116.8 (d, J = 23.2 Hz), 121.6, 122.0,
122.5, 123.7, 128.4 (d, J = 8.6 Hz), 135.1 (d, J = 13.2 Hz), 135.7 (d,
J = 13.7 Hz), 137.2 (d, J = 9.6 Hz), 141.7, 145.2, 148.1, 148.8, 152.9,
153.9 (d, J = 245.5 Hz), 161.3, 162.4, 162.7 (d, J = 249.1 Hz), 163.3,
163.9, 170.4; MS-EI m/z: 612 (M+); HR-MS (EI) m/z: 612.1279
(calcd for C32H22F2N4O5S: 612.1279).
C24H14F2N4O3S: C, 58.94; H, 3.17; N, 11.46. Found: C, 58.82; H,
2.78; N, 11.16.
4.1.3. N-(3-fluoro-4-(5-phenylthieno[2,3-d]pyrimidin-4-yloxy)
phenyl)-1-(4-fluoro phenyl)-2-oxo-1,2-dihydropyridine-3-
carboxamide (6b)
4.1.7. N-(4-(5-(biphenyl-4-yl)thieno[2,3-d]pyrimidin-4-yloxy)-
3-fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydro-
pyridine-3-carboxamide (6f)
This compound was prepared as a white solid from 5f following
a procedure similar to that of preparation of 6i in 57.8% yield. 1H
NMR (300 MHz, DMSO) d: 6.69 (d, J = 6.9 Hz, 1H), 7.39 (m, 7H),
7.58 (m, 2H), 7.74 (m, 6H), 7.94 (m, 2H), 8.09 (dd, J = 1.8, 6.3 Hz,
1H), 8.55 (dd, J = 1.8, 7.2 Hz, 1H), 8.66 (s, 1H), 12.06 (s, 1H); MS-
EI m/z: 628 (M+); HR-MS (EI) m/z: 628.1374 (calcd for
This compound was prepared as a white solid from 5b following
a procedure similar to that of preparation of 6i in 33.1% yield. 1H
NMR (300 MHz, CD3Cl) d: 6.60 (t, J = 6.6 Hz, 1H), 7.06 (t,
J = 8.4 Hz, 1H), 7.27 (m, 2H), 7.39 (m, 6H), 7.61 (d, J = 6.9 Hz, 4H),
7.92 (dd, J = 2.4 Hz, J = 12.6 Hz, 1H), 8.62 (s, 1H), 8.73 (dd, J = 2.4,
7.2 Hz, 1H), 11.95 (s, 1H); 13C NMR (100 MHz, CDCl3) d: 107.3,
109.3 (d, J = 23.3 Hz), 116.0, 116.8 (d, J = 22.7 Hz), 122.0, 123.2,
123.7, 127.9, 128.4 (d, J = 8.7 Hz), 129.4, 135.0 (d, J = 13.2 Hz),
135.5, 135.8, 137.1 (d, J = 9.6 Hz), 141.6, 145.2, 153.0, 154.0 (d,
J = 245.9 Hz), 161.3, 162.4, 162.7 (d, J = 248.6 Hz), 163.2, 170.4;
MS (EI) m/z: 552 (M+); Anal. Calcd for C30H18F2N4O3S: C, 63.65;
H, 3.47; N, 9.90. Found: C, 63.80; H, 3.26; N, 9.88.
C36H22F2N4O3S: 628.1381).
4.1.8. N-(3-fluoro-4-(5-(naphthalen-2-yl)thieno[2,3-d]
pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6g)
This compound was prepared as a white solid from 5g following
a procedure similar to that of preparation of 6i in 54.4% yield. 1H
NMR (300 MHz, DMSO) d: 6.72 (t, J = 6.9 Hz, 1H), 7.41 (m, 4H),
7.58 (m, 4H), 7.85 (d, J = 8.4 Hz, 1H), 7.94 (m, 4H), 8.05 (s, 1H),
8.10 (dd, J = 1.8, 6.6 Hz, 1H), 8.22 (s, 1H), 8.58 (dd, J = 1.5, 7.5 Hz,
1H), 8.70 (s, 1H), 12.04 (s, 1H); MS-EI m/z: 602 (M+); HR-MS (EI)
m/z: 602.1217 (calcd for C34H20F2N4O3S: 602.1224).
4.1.4. N-(3-fluoro-4-(5-(4-methoxyphenyl)thieno[2,3-d]
pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6c)
This compound was prepared as a white solid from 5c following
a procedure similar to that of preparation of 6i in 65.3% yield. 1H
NMR (300 MHz, CDCl3) d: 3.82 (s, 3H), 6.59 (t, J = 7.2 Hz, 1H),
6.93 (d, J = 7.2 Hz, 1H), 7.07 (t, J = 8.4 Hz, 1H), 7.28 (m, 4H), 7.52
(d, J = 9.0 Hz, 2H), 7.60 (d, J = 4.8 Hz, 1H), 7.93 (d, J = 12.0 Hz, 1H),
8.59 (s, 1H), 8.71 (d, J = 7.2 Hz, 1H), 11.94 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 55.2, 107.2, 109.2 (d, J = 23.2 Hz), 113.2,
116.0, 116.8, 116.9, 122.0, 122.3, 123.8, 128.0, 128.4 (d,
J = 8.6 Hz), 130.5, 135.1 (d, J = 12.8 Hz), 135.5, 135.8, 137.1 (d,
J = 9.8 Hz), 141.6, 145.1, 152.9, 154.0 (d, J = 236.0 Hz), 159.3,
161.3, 162.4, 162.7 (d, J = 249.2 Hz), 163.2, 170.4; MS (EI) m/z:
582 (M+); Anal. Calcd for C31H20F2N4O4S: C, 63.91; H, 3.46; N,
9.62. Found: C, 63.57; H, 3.29; N, 9.43.
4.1.9. N-(3-fluoro-4-(5-(4-(trifluoromethyl)phenyl)thieno[2,3-d]
pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6h)
This compound was prepared as a white solid from 5h following
a procedure similar to that of preparation of 6i in 59.3% yield. 1H
NMR (300 MHz, CDCl3) d: 6.60 (t, J = 7.2 Hz, 1H), 7.08 (t,
J = 8.7 Hz, 1H), 7.27 (m, 3H), 7.41 (m, 2H), 7.45 (s, 1H), 7.62 (d,
J = 4.5 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 8.1 Hz, 2H),
7.95 (d, J = 12.3 Hz, 1H), 8.62 (s, 1H), 8.71 (d, J = 8.1 Hz, 1H),
11.97 (s, 1H); 13C NMR (100 MHz, CDCl3) d: 107.2, 109.2 (d,
J = 23.3 Hz), 116.0, 116.4, 116.8 (d, J = 22.8 Hz), 121.9, 122.7,
123.7, 124.8, 124.9, 125.4, 128.3, 129.6, 129.8 (q, J = 32.3 Hz),
134.3, 134.8 (d, J = 12.7 Hz), 135.7, 137.3 (d, J = 10.0 Hz), 139.0,
141.7, 145.1, 153.2, 153.9 (d, J = 254.0 Hz), 161.4, 162.4, 162.7 (d,
J = 248.6 Hz), 163.1, 170.5; MS (EI) m/z: 620 (M+); Anal. Calcd for
4.1.5. N-(3-fluoro-4-(5-(5-methylthiophen-2-yl)thieno[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6d)
This compound was prepared as a white solid from 5d following
a procedure similar to that of preparation of 6i in 81% yield. 1H
NMR (300 MHz, CDCl3) d: 2.49 (s, 3H), 6.60 (m, 1H), 6.72 (s, 1H),
7.28 (m, 3H), 7.42 (m, 5H), 7.61 (d, J = 5.1 Hz, 1H), 7.95 (d,
J = 12.3 Hz, 1H), 8.59 (s, 1H), 8.72 (d, J = 6.9 Hz, 1H), 11.98 (s,
1H); 13C NMR (100 MHz, CDCl3) d: 15.3, 107.2, 109.3 (d,
J = 23.2 Hz), 116.0, 116.3, 116.8 (d, J = 23.2 Hz), 121.9, 123.0,
123.8, 127.9, 128.3 (d, J = 9.1 Hz), 128.5, 133.9, 135.0, 135.7,
137.2 (d, J = 9.5 Hz), 140.4, 141.7, 145.2, 153.0, 154.1 (d,
J = 246.4 Hz), 161.4, 162.4, 162.7 (d, J = 249.1 Hz), 163.1, 170.2;
MS (EI) m/z: 572 (M+); Anal. Calcd for C29H18F2N4O3S2: C, 60.83;
H, 3.17; N, 9.78. Found: C, 60.63; H, 3.10; N, 9.67.
C31H17F5N4O3S: C, 59.57; H, 2.82; N, 8.96. Found: C, 59.59; H,
2.63; N, 8.83.
4.1.10. N-(4-(5-(3,4-difluorophenyl)thieno[2,3-d]pyrimidin-4-
yloxy)-3-fluoro phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6j)
This compound was prepared as a white solid from 5j following
a procedure similar to that of preparation of 6i in 55.4% yield. 1H
NMR (300 MHz, CDCl3) d: 6.60 (t, J = 7.2 Hz, 1H), 7.07 (t,
J = 8.4 Hz, 1H), 7.17 (t, J = 9.0 Hz, 1H), 7.31 (m, 3H), 7.40 (m, 5H),
7.62 (d, J = 6.6 Hz, 1H), 7.95 (d, J = 12.6 Hz, 1H), 8.61 (s, 1H), 8.72
(d, J = 7.2 Hz, 1H), 11.97 (s, 1H); 13C NMR (100 MHz, CDCl3) d:
107.2, 109.2 (d, J = 23.7 Hz), 116.0, 116.4, 116.7, 116.8 (d,
J = 33.3 Hz), 116.9, 118.5 (d, J = 27.8 Hz), 121.9, 123.7 (d, J =
23.2 Hz), 125.6, 128.4 (d, J = 8.7 Hz), 132.3, 133.6, 134.8 (d,
J = 13.2 Hz), 135.7, 137.3, 141.7, 145.2, 149.6 (dd, J = 247.7,
12.3 Hz), 150.1 (dd, J = 247.7, 12.3 Hz), 153.2, 153.9 (d,
J = 246.4 Hz), 161.4, 162.4, 162.7 (d, J = 248.7 Hz), 163.1, 170.3;
4.1.6. N-(4-(5-(3,4-dimethoxyphenyl)thieno[2,3-d]pyrimidin-4-
yloxy)-3-fluoro phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (6e)
This compound was prepared as a white solid from 5e following
a procedure similar to that of preparation of 6i in 64.5% yield. 1H
NMR (300 MHz, CDCl3) d: 3.85 (s, 3H), 3.90 (s, 3H), 6.59 (t,
J = 7.2 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 8.7 Hz, 1H), 7.16

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3915
MS (EI) m/z: 588 (M+); Anal. Calcd for C30H16F4N4O3S: C, 61.22; H,
2.74; N, 9.52. Found: C, 61.06; H, 2.73; N, 9.39.
J = 6.6 Hz, 1H), 7.90 (d, J = 12.3 Hz, 1H), 8.72 (d, J = 7.5 Hz, 1H),
11.96 (s, 1H); 13C NMR (100 MHz, CDCl3) d: 22.3, 22.9, 25.4, 25.5,
25.8, 107.2, 109.1 (d, J = 23.1 Hz), 115.8, 116.2, 116.8 (d,
J = 23.1 Hz), 122.0, 123.9, 127.0, 128.4 (d, J = 8.8 Hz), 134.4, 135.7,
135.8, 136.6 (d, J = 9.7 Hz), 141.6, 145.1, 163.5 (d, J = 246.2 Hz),
161.4, 161.9, 162.1, 162.4, 162.7, 169.1; MS-EI m/z: 544 (M+);
HR-MS (EI) m/z: 544.1386 (calcd for C19H22F2N4O3S: 544.1381).
4.2. N-(3-fluoro-4-(6-phenylthieno[2,3-d]pyrimidin-4-yloxy)
phenyl)-1-(4-fluoro phenyl)-2-oxo-1,2-dihydropyridine-3-
carboxamide (8)
3-Fluoro-4-(6-phenylthieno[2,3-d]pyrimidin-4-yloxy)aniline
was prepared from 4-chloro-6-phenylthieno[2,3-d]pyrimidine as a
pale yellow solid (87.6%) by following a procedure similar to that of
preparation of compound 5i. 1H NMR (300 MHz, CDCl3 + CD3OD)
dH: 6.50 (m, 2H), 7.03 (t, J = 8.4 Hz, 1H), 7.40 (m, 3H), 7.70 (m,
3H), 8.53 (s, 1H). 13C NMR (400 MHz, DMSO) d: 101.3 (d,
J = 20.8 Hz), 109.8, 113.9, 119.8, 124.2, 126.6, 128.3 (d,
J = 13.0 Hz), 129.6, 132.5, 143.0, 148.6 (d, J = 10.2 Hz), 153.2,
154.4 (d, J = 241.6 Hz), 162.8, 168.1; MS-EI m/z 337 (M+).
Carboxamide 8 was obtained from 3-fluoro-4-(6-phenylthie-
no[2,3-d]pyrimidin-4-yloxy)aniline as a white solid (52.7%) by fol-
lowing a procedure similar to that of preparation of compound 6i.
1H NMR (300 MHz, C5D5N) d: 6.53 (t, J = 7.2 Hz, 1H), 7.41 (m, 5H),
7.62 (m, 4H), 7.91 (m, 4H), 8.51 (d, J = 12.6 Hz, 1H), 8.78 (m, 2H),
12.53 (s, 1H). 13C NMR (400 MHz, CF3COOD) d: 112.7, 113.8 (d,
J = 22.7 Hz), 114.8, 119.5 (d, J = 23.6 Hz), 121.1, 121.6, 125.4,
125.5, 128.2, 128.8, 129.9, 130.3 (d, J = 8.8 Hz), 131.5, 131.7,
132.3, 133.4, 136.2, 136.7, 138.5 (d, J = 13.8 Hz), 146.1, 149.7,
152.5, 155.8 (d, J = 249.0 Hz), 158.7, 165.4, 166.3 (d, J = 250.8 Hz),
166.6, 166.9; MS-EI m/z 552 (M+); Anal. Calcd for
4.5. N-(3-fluoro-4-(6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidin-4-yloxy)phenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (10c)
This compound was prepared as a white solid (62.0%) by follow-
ing a procedure similar to that of preparation of compound 6i. 1H
NMR (300 MHz, CDCl3) d: 1.78 (m, 4H), 1.92 (m, 2H), 2.95 (t,
J = 5.4 Hz, 2H), 3.31 (t, J = 5.4 Hz, 2H), 3.78 (s, 2H), 6.60 (t,
J = 5.4 Hz, 1H), 7.36 (m, 6H), 7.61 (dd, J = 2.1, 6.6 Hz, 1H), 7.95
(dd, J = 2.1, 12.0 Hz, 1H), 8.42 (s, 1H), 8.73 (dd, J = 2.1, 7.2 Hz,
1H), 11.98 (s, 1H); 13C NMR (100 MHz, CDCl3) d: 26.9, 27.3, 28.6,
30.1, 32.1, 107.2, 109.2 (d, J = 23.6 Hz), 116.0, 116.8 (d,
J = 23.2 Hz), 119.8, 122.0, 123.9, 128.4 (d, J = 8.8 Hz), 132.5, 135.4
(d, J = 13.0 Hz), 135.8, 137.1 (d, J = 9.7 Hz), 140.4, 141.6, 145.2,
151.5, 154.2 (d, J = 246.6 Hz), 161.3, 162.4, 162.7 (d, J = 249.0 Hz),
167.2; MS (EI) m/z: 544 (M+); Anal. Calcd for C29H22F2N4O3S: C,
63.75; H, 4.10; N, 10.25. Found: C, 64.03; H, 4.07; N, 10.28.
4.6. General procedure for synthesis of compounds 15a–f
C
30H18F2N4O3Sꢁ0.3H2O: C, 64.58; H, 3.36; N, 10.04. Found: C,
64.54; H, 3.40; N, 9.96.
4.6.1. N-(3-fluoro-4-(5-methylfuro[2,3-d]pyrimidin-4-
yloxy)phenyl)-1-(4-fluoro phenyl)-2-oxo-1,2-dihydropyridine-
3-carboxamide (15a)
4.3. N-(3-fluoro-4-(6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (10a)
This compound was obtained from 3-fluoro-4-(5-methylf-
uro[2,3-d]pyrimidin-4-yloxy)aniline as a white solid (61.0%) by fol-
lowing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, DMSO) d: 2.34 (s, 3H), 6.71 (t, J = 6.9 Hz, 1H), 7.41 (m,
4H), 7.58 (m, 2H), 7.95 (m, 2H), 8.08 (m, 1H), 8.44 (s, 1H), 8.57
(m, 1H), 12.07 (s, 1H); 13C NMR (100 MHz, DMSO) d: 9.4, 105.3,
107.2, 108.3 (d, J = 23.6 Hz), 113.9, 116.2 (d, J = 23.1 Hz), 120.2,
124.6, 129.4 (d, J = 8.8 Hz), 134.7, 134.9, 136.3, 137.3 (d,
J = 9.2 Hz), 141.4, 144.3, 145.2, 152.7, 153.6 (d, J = 244.3 Hz),
161.8, 161.9, 162.5 (d, J = 235.5 Hz), 168.5; MS (EI) m/z: 474
(M+); Anal. Calcd for C25H16F2N4O4: C, 62.70; H, 3.47; N, 11.70.
Found: C, 62.61; H, 3.16; N, 11.56.
The precursor 3-fluoro-4-(6,7-dihydro-5H-cyclopenta[4,5]thie-
no[2,3-d]pyrimidin-4-yloxy)aniline was obtained from 9a as a
white solid (93.2%) by following a procedure similar to that of
preparation of 5i. 1H NMR (300 MHz, CDCl3) d: 2.52 (m, 2H), 3.05
(t, J = 8.4 Hz, 2H), 3.14 (t, J = 8.4 Hz, 2H), 3.78 (s, 2H), 6.50 (t,
J = 9.6 Hz, 2H), 7.03 (t, J = 8.1 Hz, 2H), 8.48 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 27.7, 29.1, 30.0, 103.4 (d, J = 21.3 Hz), 110.6,
115.6, 124.2, 131.1 (d, J = 12.9 Hz), 136.2, 141.4, 145.7 (d,
J = 9.3 Hz), 151.9, 154.8 (d, J = 245.7 Hz), 162.5, 173.9; MS (EI)
m/z: 301 (M+). This compound was then converted to target com-
pound 10a as a white solid (52.8%) by following a procedure
similar to that of preparation of compound 6i. 1H NMR
(300 MHz, CDCl3) d: 1.78 (m, 4H), 1.92 (m, 2H), 2.95 (t, J = 5.4 Hz,
2H), 3.31 (t, J = 5.4 Hz, 2H), 6.60 (t, J = 7.2 Hz, 1H), 7.23 (m, 3H),
7.40 (m, 3H), 7.61 (dd, J = 2.1, 6.6 Hz, 1H), 7.95 (dd, J = 2.1,
12.0 Hz, 1H), 8.44 (s, 1H), 8.73 (dd, J = 2.1, 7.2 Hz, 1H), 11.98
(s, 1H); 13C NMR (100 MHz, CDCl3) d: 27.7, 29.1, 30.0, 107.2,
109.3 (d, J = 23.2 Hz), 115.6, 116.0, 116.8 (d, J = 23.2 Hz), 122.0,
123.8, 128.4 (d, J = 8.8 Hz), 135.5 (d, J = 12.9 Hz), 135.8, 136.1,
137.1 (d, J = 9.7 Hz), 141.7 (d, J = 5.1 Hz), 145.2, 151.7, 154.2 (d,
J = 246.6 Hz), 161.4, 162.1, 162.4, 162.7 (d, J = 249.0 Hz), 174.1;
MS (EI) m/z: 517 (M+); Anal. Calcd for C27H18F2N4O3S: C, 62.78;
H, 3.51; N, 10.85. Found: C, 62.79; H, 3.54; N, 10.85.
4.6.2. N-(3-fluoro-4-(5-phenylfuro[2,3-d]pyrimidin-4-
yloxy)phenyl)-1-(4-fluoro phenyl)-2-oxo-1,2-dihydropyridine-
3-carboxamide (15b)
This compound was obtained as a white solid (68.5%) by follow-
ing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, CD3Cl) d: 6.61 (t, J = 6.6 Hz, 1H), 7.26 (m, 3H), 7.40 (m,
6H), 7.63 (d, J = 6.0 Hz, 1H), 7.75 (m, 3H), 7.97 (d, J = 12.3 Hz, 1H),
8.52 (s, 1H), 8.73 (d, J = 6.0 Hz, 1H), 11.99 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 103.6, 107.3, 109.3 (d, J = 23.1 Hz), 116.0,
116.8 (d, J = 23.0 Hz), 121.5, 122.0, 123.7, 128.2, 128.4 (d,
J = 8.5 Hz), 128.5, 128.6, 130.0, 135.3 (d, J = 13.0 Hz), 135.7, 137.3
(d, J = 9.7 Hz), 139.9, 141.7, 145.2, 153.1, 154.1 (d, J = 246.7 Hz),
161.4, 162.4, 162.7 (d, J = 258.1 Hz), 163.3, 169.1; MS (EI) m/z:
536 (M+); Anal. Calcd for C30H18F2N4O4: C, 66.16; H, 3.50; N,
10.29. Found: C, 66.42; H, 3.48; N, 10.21.
4.4. N-(3-fluoro-4-(2-methyl-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (10b)
4.6.3. N-(3-fluoro-4-(5-(thiophen-2-yl)furo[2,3-d]pyrimidin-4-
yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-
carboxamide (15c)
This compound was obtained as a white solid (40.5%) by follow-
ing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, CDCl3) d: 6.61 (t, J = 6.6 Hz, 1H), 7.10 (t, J = 4.2 Hz, 1H),
This compound was prepared as a white solid (56.5%) by follow-
ing a procedure similar to that of preparation of compound 6i. 1H
NMR (300 MHz, CDCl3) d: 1.89 (m, 4H), 2.52 (s, 3H), 2.83 (s, 2H),
3.03 (s, 2H), 6.60 (t, J = 7.2 Hz, 1H), 7.30 (m, 5H), 7.61 (d,

3916
A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
7.25 (m, 4H), 7.40 (m, 3H), 7.62 (m,2H), 7.86 (s, 1H), 7.97 (dd,
J = 2.4, 12.3 Hz, 1H), 8.51 (s, 1H), 8.73 (dd, J = 1.8, 7.2 Hz, 1H),
11.99 (s, 1H); 13C NMR (100 MHz, CDCl3) d: 103.1, 107.2, 109.2
(d, J = 23.2 Hz), 115.1, 116.0, 116.8 (d, J = 23.2 Hz),121.8, 123.7,
125.4, 127.7, 127.9, 128.3 (d, J = 9.1 Hz), 130.9, 135.0 (d,
J = 13.3 Hz), 135.7, 137.4 (d, J = 10.0 Hz), 139.3, 141.7, 145.2,
153.2, 154.0 (d, J = 246.4 Hz), 161.4, 162.3, 162.6 (d, J = 254.7 Hz),
163.1, 168.8; MS (EI) m/z: 542 (M+) HR-MS (EI) m/z: 542.5128
(calcd for C28H16F2N4O4S: 542.5120).
brine, dried over Na2SO4, and concentrated under reduced pres-
sure. The crude product was purified by silica gel column chro-
matograph to afford the title compound 16 (54 mg, 78.7%) as a
white solid. 1H NMR (300 MHz, DMSO) d: 3.24 (s, 3H), 6.71 (t,
J = 7.2 Hz, 1H), 7.42 (m, 3H), 7.51 (m, 4H), 7.59 (m, 2H), 7.81 (d,
J = 7.2 Hz, 2H), 8.02 (dd, J = 2.4, 12.6 Hz, 1H), 8.10 (dd, J = 2.4,
6.9 Hz, 1H), 8.56 (dd, J = 1.8, 7.5 Hz, 1H), 8.72 (s, 1H), 12.12 (s,
1H); 13C NMR (100 MHz, CDCl3) d: 39.1, 105.6, 107.1, 108.0,
116.1 (d, J = 25.5 Hz), 120.1, 120.9, 124.3, 128.6, 128.8, 129.0,
129.3 (d, J = 8.7 Hz), 134.0 (d, J = 12.8 Hz), 136.3, 137.6 (d,
J = 9.6 Hz), 144.2, 144.7, 144.9, 153.1 (d, J = 243.7 Hz), 159.0,
161.7, 161.8, 161.9 (d, J = 244.6 Hz), 162.9, 168.0; MS (EI) m/z:
614 (M+); HR-MS (EI) m/z: 614.1068 (calcd for C31H20F2N4O6S:
614.1072).
4.6.4. N-(3-fluoro-4-(2-methyl-5-(thiophen-2-yl)furo[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (15d)
This compound was obtained as a white solid (67.4%) by follow-
ing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, CDCl3) d: 2.57 (s, 3H), 6.61 (t, J = 6.9 Hz, 1H), 7.08 (m,
1H), 7.26 (m, 4H), 7.37 (m, 3H), 7.64 (m, 2H), 7.77 (s, 1H), 7.94
(dd, J = 2.4, 12.3 Hz, 1H), 8.73 (dd, J = 1.8, 7.2 Hz, 1H), 11.97 (s,
1H); 13C NMR (100 MHz, CDCl3) d: 25.5, 100.2, 107.3, 109.1 (d,
J = 22.8 Hz), 115.0, 115.8, 116.8 (d, J = 22.8 Hz), 121.9, 123.8,
125.1, 127.5, 127.9, 128.3 (d, J = 8.7 Hz), 131.4, 135.3 (d,
J = 13.2 Hz), 135.7, 136.9 (d, J = 10.0 Hz), 138.5, 141.7, 145.2,
154.2 (d, J = 246.4 Hz), 161.4, 162.4, 162.5, 162.7 (d, J = 253.7 Hz),
163.8, 169.5; MS (EI) m/z: 556 (M+). HR-MS (EI) m/z: 556.5394
(calcd for C29H18F2N4O4S: 556.5396.
4.8. N-(3-fluoro-4-(2-(methylamino)-5-phenylfuro[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (17)
To a solution of carboxamide 16 (44 mg, 0.072 mmol) and
methylamine hydrochloride (9.7 mg, 0.143 mmol) in 1,4-dioxane
(2 mL), was added DIPEA (27.9 mg, 0.216 mmol). The solution
was irradiated for 1 h with microwave at 140 °C. The solvent was
removed under reduced pressure and the residue was diluted with
water and EtOAc. The organic layer was separated, washed with
brine, dried over Na2SO4, and concentrated under reduced pressure
to give a crude product, which was purified by silica gel column
chromatograph to afford compound 17 (10 mg, 24.7%) as a white
solid. 1H NMR (300 MHz, CDCl3) d: 2.89 (s, 3H), 4.99 (q,
J = 5.1 Hz, 1H), 6.60 (t, J = 6.3 Hz, 1H), 7.14 (t, J = 8.4 Hz, 1H), 7.24
(m, 1H), 7.33 (m, 2H), 7.40 (m, 6H), 7.61 (m, 1H), 7.73 (m, 2H),
7.88 (dd, J = 2.4, 12.0 Hz, 1H), 8.74 (dd, J = 1.2, 12.0 Hz, 1H), 11.92
(s, 1H); MS (EI) m/z: 565 (M+); HR-MS (EI) m/z: 565.1567 (calcd
for C31H21F2N5O4: 565.1562).
4.6.5. N-(3-fluoro-4-(2-(methylthio)-5-phenylfuro[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (15e)
This compound was obtained as a white solid (43.7%) by follow-
ing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, CDCl3) d: 2.35 (s, 3H), 6.59 (t, J = 5.4 Hz, 1H), 7.22 (m,
4H), 7.38 (m, 5H), 7.60 (m, 2H), 7.73 (d, J = 10.5 Hz, 2H), 7.91 (d,
J = 10.5 Hz, 1H), 8.72 (d, J = 7.2 Hz, 1H), 11.98 (s, 1H); 13C NMR
(100 MHz, CDCl3) d: 14.2, 99.4, 107.2, 108.8 (d, J = 23.7 Hz),
115.6, 116.7 (d, J = 23.2 Hz), 121.4, 121.8, 123.8, 128.0, 128.3,
128.6, 130.1, 135.2 (d, J = 12.8 Hz), 135.7, 137.0 (d, J = 9.2 Hz),
138.2, 141.7, 145.1, 154.1 (d, J = 246.0 Hz), 161.4, 162.1, 162.4,
162.6 (d, J = 252.8 Hz), 167.2, 169.8; MS (EI) m/z: 582 (M+). HR-
MS (EI) m/z: 582.5767 (calcd for C31H20F2N4O4S: 582.5780).
4.9. N-(4-(2-azido-5-phenylfuro[2,3-d]pyrimidin-4-yloxy)-3-
fluorophenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-
3-carboxamide (18)
To a solution of methylsulfone 17 (25 mg, 0.041 mmol) in DMF
(5 mL) was added NaN3 (27.9 mg, 0.216 mmol) at 0 °C. The mixture
was allowed to warm to rt and was stirred for 1.5 h. The reaction
was quenched with water (10 mL) and the mixture was extracted
with EtOAc (3 ꢄ 10 mL). The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The crude product
was purified by column chromatograph to give azide 18 (7 mg,
30.4%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) d: 6.60
(t, J = 6.9 Hz, 1H), 7.19 (m, 1H), 7.26 (m, 2H), 7.41 (m, 5H), 7.61
(m, 2H), 7.71 (m, 2H), 7.92 (dd, J = 2.7, 12.3 Hz, 1H), 8.73 (dd,
J = 2.1, 6.9 Hz, 1H), 11.97 (s, 1H); MS (EI) m/z: 577 (M+); HR-MS
(EI) m/z: 577.1291 (calcd for C30H17F2N7O4: 577.1310).
4.6.6. N-(3-fluoro-4-(5-(4-fluorophenyl)furo[2,3-d]pyrimidin-4-
yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-
3-carboxamide (15f)
This compound was obtained as a white solid (49.2%) by follow-
ing a procedure similar to that of preparation of 6i. 1H NMR
(300 MHz, CDCl3) d: 6.61 (t, J = 7.2 Hz, 1H), 7.14 (m, 3H), 7.23 (m,
2H), 7.41 (m, 3H), 7.62 (m, 1H), 7.71 (m, 3H), 7.97 (dd, J = 2.4,
12.0 Hz, 1H), 8.52 (s, 1H), 8.73 (dd, J = 2.1, 7.5 Hz, 1H), 11.99 (s,
1H); 13C NMR (100 MHz, CDCl3) d: 103.6, 107.3, 109.3 (d,
J = 23.1 Hz), 115.7 (d, J = 21.3 Hz), 116.0, 116.8 (d, J = 23.2 Hz),
120.6, 122.0, 123.7, 126.0, 128.4 (d, J = 8.8 Hz), 130.3 (d,
J = 7.8 Hz), 135.2 (d, J = 13.0 Hz), 135.7, 137.3 (d, J = 9.9 Hz),
139.7, 141.7, 145.2, 153.2, 154.0 (d, J = 246.2 Hz), 161.4, 162.4,
162.7 (d, J = 250.4 Hz), 162.8 (d, J = 246.2 Hz), 163.2, 169.1; MS
(EI) m/z: 554 (M+); HR-MS (EI) m/z: 554.1203 (calcd for
4.10. N-(3-fluoro-4-(2-methoxy-5-phenylfuro[2,3-d]pyrimidin-
4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (19)
C
30H17F3N4O4: 554.1202).
To a solution of NH3 in CH3OH was added methylsulfone 16
(32 mg, 0.052 mmol). The mixture was stirred overnight at rt.
The solvent was evaporated under reduced pressure and the resi-
due was poured into water and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The crude product was puri-
fied by silica gel column chromatograph to afford 2-methoxypyr-
imidine 19 (13 mg, 44.0%) as a white solid. 1H NMR (300 MHz,
CDCl3) d: 3.89 (s, 3H), 6.60 (t, J = 6.3 Hz, 1H), 7.19 (m, 1H), 7.25
(m, 2H), 7.40 (m, 6H), 7.62 (m, 2H), 7.73 (d, J = 7.8 Hz, 2H), 7.92
4.7. N-(3-fluoro-4-(2-(methylsulfonyl)-5-phenylfuro[2,3-
d]pyrimidin-4-yloxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide (16)
To a solution of carboxamide 15e (65 mg, 0.112 mmol) in
CH2Cl2 (5 mL), was added mCPBA (57.6 mg, 0.335 mmol) at 0 °C.
The mixture was stirred for 2.5 h, and then quenched with NaHCO3
saturated solution. The organic layer was separated, washed with

A. Zhao et al. / Bioorg. Med. Chem. 19 (2011) 3906–3918
3917
(d, J = 12.0 Hz, 1H), 8.73 (d, J = 6.9 Hz, 1H), 11.95 (s, 1H); MS (EI)
m/z: 566 (M+); HR-MS (EI) m/z: 566.1399 (calcd for C31H20F2N4O5:
566.1402).
ip administration 7 days per week for 3 weeks. The sizes of the
tumors were measured twice per week using microcaliper. The
tumor volume (TV) was calculated as: TV = (length ꢄ width2)/2.
Tumor volume was shown on indicated days as the median tumor
volume SE indicated for groups of mice. Percent (%) inhibition
values were measured on the final day of study for drug-treated
compared with vehicle-treated mice and are calculated as
100 ꢄ {1ꢂ[(treated final day-treated day 1)/(control final day-
control day 1)]}. Significant differences between the treated
versus the control groups (P 6 0.001) were determined using
Student’s t-test.
4.11. Pharmacological procedure
4.11.1. ELISA kinase assay
The tyrosine kinase activities were evaluated according to the
reported protocol. Briefly, in enzyme-linked-immunosorbent assay
(ELISA), 20
lg/mL Poly(Glu,Tyr) 4:1 (Sigma) was pre-coated as a
substrate in 96-well plates. Fifty microliters of 10
lM ATP solution
diluted in kinase reaction buffer (50 mM HEPES pH 7.4, 50 mM
MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added to
Acknowledgments
each well. Various concentrations of compounds diluted in 10 lL
of 1% DMSO (v/v) were added to each reaction well, with 1% DMSO
(v/v) used as the negative control. The kinase reaction was initiated
by the addition of purified tyrosine kinase proteins diluted in 40 lL
of kinase reaction buffer solution. After incubation for 60 min at
37 °C, the plate was washed three times with Phosphate Buffered
Saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 lL anti-
phosphotyrosine (PY99) antibody (1:500 diluted in 5 mg/mL BSA
T-PBS) was added. After 30 min incubation at 37 °C, the plate
was washed three times. Hundred microliters horseradish peroxi-
dase-conjugated goat anti-mouse IgG (1:2000 diluted in 5 mg/mL
BSA T-PBS) was added. The plate was reincubated at 37 °C for
The authors are grateful to Shanghai Commission of Science and
Technology (10JC1417100), National Science & Technology Major
Project on ‘Key New Drug Creation and Manufacturing Program’
(2009ZX09301-001, 2009ZX09103-062), the Natural Science
Foundation of China for Distinguished Young Scholars (No.
30725046), and Program of Shanghai Subject Chief Scientist (No.
10XD1405100).
Supplementary data
Supplementary data (copies of 1H and 13C NMR data for all the
new compounds as well as the key intermediates) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2011.05.038.
30 min, and washed as before. Finally, 100 lL of a solution contain-
ing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate
buffer, pH 5.5, was added and samples were incubated at room
temperature until color emerged. The reaction was terminated by
the addition of 50 lL of 2 M H2SO4, and the plate was read using
References and notes
a multiwell spectrophotometer (VERSAmax™, Molecular Devices,
Sunnyvale, CA, USA) at 490 nm. The inhibition rate (%) was calcu-
lated using the following equation: ½1 ꢂ ðA490=A490 controlÞꢃꢄ
100%. IC50 values were calculated from the inhibition curves.
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