Design, synthesis and biological evaluation of novel pyrimidine, 3-cyanopyridine and m-amino-N-phenylbenzamide based monocyclic EGFR tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.03.053

36 new compounds with the typical skeleton of 4-anilino-5-vinyl/ethynyl pyrimidine, 4-anilino-3-cyano-5-vinyl/ethynyl/phenyl pyridine, and m-amino-N-phenylbenzamide, are designed, synthesized and selectively tested on EGFR, ErbB-2 kinases, and A-549, HL60 cells growth inhibition. Results from the bioactivity and chemical structures yield preliminary structure-activity relationships (SARs). The most potent 5-ethynylpyrimidine derivative 20a has an IC₅₀ value of 45 nM to EGFR kinase. Several compounds of other series also show IC₅₀ values <1 μM for EGFR and <5 μM for A-549 and HL60 cells growth inhibition.

Full text of DOI:10.1016/j.bmc.2013.03.053

Bioorganic & Medicinal Chemistry 21 (2013) 3090–3104
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel pyrimidine,
3-cyanopyridine and m-amino-N-phenylbenzamide based
monocyclic EGFR tyrosine kinase inhibitors
Yongjun Mao a, Wenxiu Zhu b, Xiaoguang Kong b, Zhen Wang a, Hua Xie a, Jian Ding a,
Nicholas Kenneth Terrett b, Jingkang Shen a, Jingshan Shen a,
⇑
a Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Pudong, Shanghai 201203, China
b Vitargeta Therapeutics Inc., Plainsboro, NJ 08536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
36 new compounds with the typical skeleton of 4-anilino-5-vinyl/ethynyl pyrimidine, 4-anilino-3-
cyano-5-vinyl/ethynyl/phenyl pyridine, and m-amino-N-phenylbenzamide, are designed, synthesized
and selectively tested on EGFR, ErbB-2 kinases, and A-549, HL60 cells growth inhibition. Results from
the bioactivity and chemical structures yield preliminary structure–activity relationships (SARs). The
most potent 5-ethynylpyrimidine derivative 20a has an IC50 value of 45 nM to EGFR kinase. Several com-
Received 30 January 2013
Revised 9 March 2013
Accepted 19 March 2013
Available online 1 April 2013
pounds of other series also show IC50 values <1
inhibition.
l
M for EGFR and <5
l
M for A-549 and HL60 cells growth
Keywords:
EGFR
5-Ethynylpyrimidine
4-Anilino-3-cyano-5-phenylpyridine
m-Amino-N-phenylbenzamide
Tyrosine kinase inhibitors
Ó 2013 Published by Elsevier Ltd.
1. Introduction
EGFR inhibition.5 The third one lapatinib is an equipotent inhibitor
of both EGFR and ErbB-2, which has been expected to provide bet-
ter efficacy than single receptor targeting.6 The chemical structures
are shown in Figure 1. Among the known kinase inhibitors, the
4-anilinoquinazoline scaffold is the most common template for
inhibitors of the EGFR family.7 In the last few years, a large struc-
tural variety of compounds, such as 4-anilino-3-cyanoquinolines
(neratinib),8 4-anilinopyrazolo[3,4-d]pyrimidines,9 4-anilinopyraz-
olo and 4-anilinopyrroloquinazolines,10 were reported as EGFR
tyrosine kinase inhibitors.
In our effort to develop new non-quinazoline derivatives as
effective EGFR kinase inhibitors, we focused on the monocyclic
scaffolds. Several series of structures were designed, including
4-anilino-5-vinyl and 5-ethynylpyrimidines (16, 20), 3-cyano-4-
anilino-5-vinyl and 5-ethynylpyridines (29, 33), 3-cyano-4-
anilino-5-phenylpyridines(35),andm-amino-N-phenylbenzamides
(48–50,61),asdepictedinTables1–4.Wehopethe6-methylpyrimidine
scaffold, the 3-cyano-6-methylpyridine scaffold, or even the simple
5-amido-4-ethoxybenzamide structure can mimic the function
of the classic quinazoline or 3-cyanoquinoline moiety and are
expected to offer better physicochemical properties.11
The epidermal growth factor receptor (EGFR/ErbB-1) and the re-
lated human epidermal growth factor receptor-2 (ErbB-2/Her-2)
belong to the family of trans-membrane growth factor receptor
protein tyrosine kinases (PTKs), which play critical roles in many
of the signal transduction processes that control cell growth, differ-
entiation, mitosis and apoptosis.1 The ErbB receptors can be acti-
vated through homo or heterodimerization with other receptors
resulting in phosphorylation events and downstream signaling
that produce excessive growth by inducing cell proliferation and
inhibiting apoptotic pathways.2 Overexpression of these receptors
is found in a variety of cancers such as breast, ovarian, colon and
non-small-cell lung cancers (NSCLC), and has been associated with
poor prognosis in patients.3 In addition, coexpression of EGFR and
Her-2 has been found in various cancers such as breast, ovarian,
colon and prostate cancers, and is associated with poor prognosis
of the patients. Therefore, simultaneous inhibition of EGFR and
Her-2 is expected to provide superior efficacy to single receptor
targeting.4
Over the past decades, there are two clinically approved 4-ani-
linoquinazoline inhibitors, gefitinib and erlotinib, selective for
Six designed compounds were selected and overlaid with lapat-
inib and neratinib respectively before the synthesis work, as
depicted in Figures 2 and 3. PyMOL software was used to align
the compounds. Lapatinib and compounds 16b, 20a and 33a were
⇑
Corresponding author.
E-mail address: jsshen@mail.shcnc.ac.cn (J. Shen).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.03.053

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3091
thought that the linker in our pyrimidine series should be in sim-
ilar orientation as the phenyl ring of quinazoline, that is, a substi-
tuent at the C-5 position was required to be coplanar with the
pyrimidine ring. Thus, we tried to find a linker that could maintain
coplanarity with the pyrimidine nucleus. Similar idea was used for
design the 5-substituted-3-cyanopyridine from 3-cyanoquinoline.
The eight compounds 16, 20, 29 and 33 were designed and syn-
thesized, as depicted in Schemes 1–3, and tested on EGFR and
ErbB-2 kinase inhibition activities. The IC50 values are shown in Ta-
ble 1. In the same assay neratinib has an IC50 value of 2.2 nM.13 The
most potent one among the eight is 20a (IC50 = 45 nM) which has
the 5-ethynylpyrimidine skeleton with 3-chloro-4-(3-fluoroben-
zyloxy)anilino substituent at the 4-position. The 5-vinylpyrimidine
16b also has an IC50 value <1 lM, which has the best overlay re-
sults with lapatinib, as shown in Figure 2. Other derivatives show
no interesting bioactivities, especially to ErbB-2 kinase that most
of them have poor inhibition value.
Another series of 3-cyano-4-anilino-5-phenylpyridines 35 were
designed and synthesized (Scheme 4). The structures and bioassay
data are depicted in Table 2. A 4-substituted phenyl ring was intro-
duced to the 5-position to extend the conformation of the mole-
cule. The 4-anilino chains of lapatinib and neratinib were
introduced to the 4-position respectively. While this modification
was not successful, only 35c has an IC50 value of 0.6 lM on EGFR
Figure 1. Chemical structure of compounds 1–4.
and 35d has some inhibition activities on the cells growth, which
indicated that the 3-cyano-5-phenylpyridine skeleton is not potent
as EGFR kinase inhibitor.
imported into PyMOL, and optimized the dynamics conformation
respectively. Then the 3 compounds were overlaid on lapatinib
manually to give the image in Figure 2. The same process was used
to give Figure 3. Except for 33a, other five can give good to medium
overlap results.
The 5-alkenyl and alkynyl substituents can be coplanar with the
pyrimidine or 3-cyanopyridine ring, these scaffolds should be able
to effectively mimic the well-known 4-anilinoquinazoline or
3-cyanoquizoline kinase inhibitor template. The phenyl ring as the
C-5 substituent (35a) was introduced to the 3-cyanopyridine ring
to mimic the 3-cyanoquizoline template. 2-Nitro-N-phenylbenza-
mides (48a) and 2-methyl-N-phenylbenzamides (61a) were also
designed to mimic the 4-anilinoquinazoline and 3-cyanoquizoline
template, as shown in Figure 4.12
In this paper, we will report the synthesis, bioactivity assay and
a primary structure–activity relationships (SARs) study of the
above monocyclic skeleton as inhibitors of EGFR kinase. The new
3-cyanopyridine and pyrimidine compounds were tested respec-
tively on EGFR and ErbB-2 kinases inhibition activities, some com-
pounds were also selected to test in a carcinoma cell growth
inhibition assay on two cell lines, A-549 and HL60. The IC50 data
of the tyrosine kinases inhibition or the cell viability assay were gi-
ven. The SARs were provided based on the biological activity for
these inhibitors of the EGFR signaling.
The other part of our work is design the 5-amido-4-ethoxybenz-
amide compounds 48–50 (Schemes 5 and 6) and the 2-methyl
substituted analogues 61 (Scheme 7), with the purpose of unfold-
ing the N-heterocyclic ring and keeping the phenyl ring of the clas-
sic quinazoline or 3-cyanoquinoline inhibitors. Therefore the side
chains of lapatinib and neratinib, as well as the moiety developed
by us previously, were introduced to the related positions in our
new designed molecules. The structures and biological data are
shown in Tables 3 and 4, while in the present situation, no exciting
results were obtained. With regard to the EGFR kinase inhibition
assay, several derivatives show <1 lM IC50 value, including com-
pounds 48a, 48b, 49a and 50a, which has the 3-chloro-4-(pyri-
din-2-ylmethoxy)anilino substituent in the corresponding
position. The 2-nitro substituted compounds 48 can have better
inhibition activity than the related 2-amino compounds 49 on
the cells growth assay. Only two kinds of side chains were investi-
gated at the 5-position, (dimethylamino)but-2-enamide and
[(4-methylpiperazin-1-yl)methyl]benzamide. In general, the for-
mer analogues can possess better bioactivities than the latter.
Lastly four 2-methyl-5-amidobenzamides 61 were synthesized
which have the similar bioassay results to compounds 48–50.
The 3-chloro-4-(3-fluorobenzyloxy)anilino substituted 61a and
61b have ꢀ0.5
lM IC50 value, while most of these derivatives show
no high bioactivities to inhibition of the cells growth.
2. Results and discussion
2.1. Structure–activity relationships
2.2. Chemical synthesis
On the basis of the existed structureÀactivity relationship (SAR)
information,11,12 we designed pyrimidine or 3-cyanopyridine
based analogues with a 5-vinyl/ethynyl/phenyl moiety at the C5
position and bulky aniline moiety at the C4 position to secure irre-
versible EGFR inhibitory activity. m-Amino-N-phenylbenzamides
were also designed to mimic the 4-anilinequinazoline core.
Our effort is to discover and develop non-quinazoline or non-3-
cyanoquinoline derivatives for effective EGFR inhibitors as antitu-
mor agents, we focused on the pyrimidine, 3-cyanopyridine, and
m-amino-N-phenylbenzamide scaffolds which could mimic the
function of the quinazoline and 3-cyanoquinoline moiety and
could be expected to offer better physicochemical properties. We
The 4-anilino-5-vinylpyrimidines 16 and 5-ethynylpyrimidines
20 were synthesized, as shown in Schemes 1 and 3. The side chain
compounds 7 and 8 were prepared using the known method.14 The
key 4-chloropyrimidine intermediate 12 was prepared from ethyl
acetoacetate and thiourea based on the simple route,15 which cou-
pled with 3-chloro-4-(3-fluorobenzyloxy)aniline (13) to produce
the compound 14 in good yield. Using the Stille coupling,16 inter-
mediate 15 was obtained, which reacted with 7 and 8 respectively
under the Heck reaction condition17 to give the final 5-vinyl prod-
ucts 16. Base on the similar method, 5-ethynyl compounds 20 were
produced. A different 3-chloro-4-(pyridin-2-ylmethoxy)phenoxy
substituent was introduced to the 4-position.

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Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
Table 1
Biological activity data of compounds 16, 20, 29 and 33
a
Compd
Structure
IC50 (lM)
EGFR
>5
ErbB2
>10
16a
16b
0.46
>10
20a
20b
29a
0.045
>10
>5
—
>5
>10
29b
33a
33b
>5
1.5
>5
>10
—
—
a
The IC50 values are the average of three separate experiments. Standard deviations were below 30%.
The 3-cyanopyridine derivatives 29 and 33 were synthesized
The synthetic route of 35 is shown in Scheme 4. 2-(4-Nitro-
phenyl)acetic acid was converted to the 3-oxobutanenitrile 37
through three simple steps, which then reacted in turn with
DMF–DMA and veratrylamine to give the intermediate 39 based
on the similar reported method.18 The key 4-chloroquinoline 40
was obtained after the chloration. Compounds 35 were produced
using the similar method (Schemes 2 and 3). The key 4-chloro-3-
cyanopyridine intermediate 25 was prepared from the 4-hydroxy
nicotinic acid 22 through the amidation, iodosubstitution and chl-
oration. Adopted the same method to preparation of 16 and 20, 29
and 33 were obtained.

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3093
Table 2
Chemical structure and biological activity data of compounds 35
R1
R2
R3
IC50 (l
M)
Cell growth inhibition
HL60
a
Compd
EGFR
>5
A-549
>10
35a
–Cl
>10
35b
35c
35d
–Cl
–Cl
–Cl
2.3
>10
7.0
6.0
6.0
>10
1.8
0.66
2.5
35e
35f
–F
–Cl
–Cl
–H
–H
3.5
3.0
>5
>10
>10
>10
>10
6.0
5.0
>10
5.0
–F
35g
35h
-COMe
-COMe
>5
a
The IC50 values are the average of three separate experiments. Standard deviations were below 30%.
by another two steps using the similar method described
previously.
and cells growth, probably because the bulky 5-phenyl substituent
transfers the molecular conformation too much to lose the bioac-
tivities, except for the [(N-methyl piperazin-1-yl)methyl]benzam-
ide substituted compounds (35d and 35f) can have moderate
inhibition to HL60 proliferation. (3) The m-aminobenzamides also
have no interesting bioactivities to EGFR or to cells, which indi-
cates that the pyrimidine core in the quinazoline, and the 3-cyano-
pyridine ring in the 3-cyanoquinaline are critical to keep the
bioactivities.
The synthetic method of 48 and 49 are depicted in Scheme 5.
The materials 51 and 52 were prepared by our improved method,19
which reacted with the anilines 13, 26 or 41, then went by reduc-
tion and amidation, compounds 48 and 49 was obtained. Using an-
other material 55, derivatives 50 were produced in the similar way
(Scheme 6). Compounds 61 were also prepared by the similar
method (Scheme 7).
3. Conclusion
4. Experimental
In summary, 36 new compounds derived from four series of the
monocyclic scaffolds, 5-vinyl/ethynyl pyrimidine (16, 20), 3-cya-
no-5-vinyl/ethynyl pyridine (29, 33), 3-cyano-5-phenylpyridine
(35), and m-amino-N-phenylbenzamide (48–50, 61), were de-
signed, synthesized and tested respectively on EGFR and ErbB-2 ki-
nases inhibition activities, most of the derivatives were also tested
in a carcinoma cell growth inhibition assay on A-549 and HL60 cell
lines. The biological activity results, the chemical structures, as
well as the overlay processing of the representative compounds
with lapatinib or neratinib, identified preliminary SARs. (1) 4-Ani-
lino-5-vinyl/ethynyl pyrimidines have better activities to EGFR ki-
nases than the similar 3-cyanopyridines. 5-Ethynyl substituted
compounds are more potent than the 5-vinyl compounds, probably
because the former have a similar molecular conformation to the
typical 4-anilinoquinazoline inhibitors. The novel 5-[(furan-2-
yl-)ethynyl]pyrimidine compound 20a has a moderate inhibitory
activity against EGFR kinases (IC50 = 45 nM). (2) The 3-cyano-5-
phenylpyridines have poor inhibitory activities to EGFR kinases
4.1. Materials and methods
All commercially available materials and solvents were used as
received without any further purification. 1H and 13C NMR spectra
were recorded with a Gemini-300 spectrometer using TMS as an
internal standard. The mass spectra were obtained from a Finnigan
MAT-95/711 spectrometer. Melting points were measured on a
Buchi-510 melting point apparatus and was uncorrected. The HPLC
results were generated using a Waters 2487 UV/Visible Detector
and Waters 515 Binary HPLC Pump.
4.2. Chemistry
4.2.1. N-(3-Bromophenyl)-4-(dimethylamino)but-2-enamide
(7)
4-(Dimethylamino)but-2-enoic acid hydrobromide
2.4 mmol) and DMF (9 L, 5 mol %) were added into THF (8 mL)
and the suspension was cooled in an ice-water bath under N2
6 (0.5 g,
l

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Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
Table 3
Chemical structure and biological activity data of compounds 48–50
a
Compd
R1
R2
R3
IC50
(
l
M)
Cell growth inhibition
HL60
EGFR
0.85
A-549
48a
49a
48b
49b
–NO2
–NH2
–NO2
–NH2
2.5
>10
3.5
6.0
4.0
>10
5.0
8.0
0.80
0.90
1.0
48c
49c
48d
49d
–NO2
–NH2
–NO2
–NH2
2.0
1.5
5.5
5.0
3.5
5.5
6.0
6.0
4.0
6.0
4.5
7.0
48e
49e
48f
49f
–NO2
–NH2
–NO2
–NH2
6.0
>10
>10
6.0
6.0
4.5
5.0
>10
5.5
0.80
>10
>10
50a
–H
1.2
>10
>10
50b
50c
50d
–H
–H
–H
3.5
6.0
6.0
>10
>10
>10
>10
>10
a
The IC50 values are the average of three separate experiments. Standard deviations were below 30%.
atmosphere. Oxalyl chloride (200
wise and the reaction suspension was stirred at 26–30 °C for
1.5 h, then cooled again in ice-water bath. 3-Bromoaniline
(175 lL, 1.6 mmol) was added dropwise and the reaction suspen-
l
L, 2.3 mmol) was added drop-
sion was stirred at 0 °C for another 4 h. The reaction solution was
diluted with water and adjusted to pH ꢀ9 with 10% NaOH solution,
extracted with EtOAc. The organic layer was washed with water
and brine, and dried over anhydrous Na2SO4. The solvent was

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3095
Table 4
Chemical structure and biological activity data of compounds 61
R1
R2
IC50 (lM)
a
Compd
Cell growth inhibition
HL60
EGFR
0.45
A-549
7.0
61a
61b
61c
61d
5.5
6.0
—
0.60
5.0
5.5
>10
>10
6.0
—
a
The IC50 values are the average of three separate experiments. Standard deviations were below 30%.
Figure 2. Overlay of lapatinib with compounds 16b, 20a and 33a. Lapatinib is
colored yellow. Compound 16b is shown in pink, 20a is green, and 33a is grey.
Atoms are colored by element with nitrogen in blue, oxygen red, chlorine dark
green, sulfur yellow and fluorine cyan.
Figure 3. Overlay of neratinib with compounds 35a, 48a and 61a. Neratinib is
colored yellow. Compound 35a is shown in grey, 48a is pink, and 61a is green.
Atoms are colored by element with nitrogen in blue, oxygen red, chlorine dark
green and fluorine cyan.
removed under reduced pressure to give the crude product, which
was purified by column chromatography on silica gel (eluent: PE/
CH2Cl2 = 5/1–1/1) to give 7 (0.33 g, 75%) as a grey solid. 1H NMR
(CDCl3, d): 2.26(s, 6H), 3.12(d, 2H), 6.11(d, 1H), 6.91–7.01(m, 1H),
7.13–7.23(m, 1H), 7.46–7.54(m, 2H), 7.72(m, 1H), 7.82(s, 1H).
the resulting solution was stirred at room temperature for 2 h.
NaBH4 (0.48 g, 12.8 mmol) was added portion-wise and the solution
was stirred at room temperature for another 4 h. The reaction solu-
tion was quenched with saturated Na2CO3, extracted with CH2Cl2.
The organic layer was washed with water and brine, and dried over
anhydrous Na2SO4. The solvent was removed under reduced pres-
sure to give 8 (0.68 g, 94%) as a light-yellow oil. 1H NMR (CDCl3, d):
2.99(s, 3H), 3.13(br, 4H), 3.76(s, 2H), 6.18(d, 1H), 6.22(s, 1H).
4.2.2. N-((5-Bromofuran-2-yl)methyl)-2-(methylsulfonyl)
ethanamine (8)
2-(Methylsulfonyl)ethanamine hydrochloride (0.5 g, 3.1 mmol)
was added to a NaOMe solution, which was prepared from Na
(0.11 g, 4.78 mmol) and anhydrous MeOH (9 mL). The reaction
mixture was stirred at room temperature for 15 min. The methanol
was removed under reduced pressure and the residual was sus-
pended into CH2Cl2 (15 mL) and filtered to removed the excess salt.
The filtrate was concentrated to give 2-(methylsulfonyl)ethanamine
as a colorless oil, which was dissolved into anhydrous MeOH
(10 mL). 5-Bromo-2-furaldehyde (0.45 g, 2.6 mmol) was added and
4.2.3. 4-Chloro-5-iodo-6-methylpyrimidine (12)
Na (30 g, 1.3 mol) was added to anhydrous MeOH (500 mL)
which was cooled in an ice-water bath. After the sodium dissolved,
thiourea (40 g, 0.52 mol) and ethyl acetoacetate (80 mL, 0.6 mol)
were added and the reaction solution was stirred and heated to re-
flux for 8 h. The methanol was removed under reduced pressure
and water (600 mL) was added to dissolve the residual solid. AcOH

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Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
Ph
Ph
Z
R1
HN
X
R1
Z
A
N
B
H
A
N
B
H
H
H
H
H
O
F
O2S
16, 20
29, 33
NH
HN
Cl
X = NH, O
Z = N, C-CN
O
N
O
A
N
B
N
O2S
R1
=
NH
HN
O
lapatinib
R1
HN
O
R1
O
Ph
R1
HN
O
Ph
O
HN
Ph
N
HN
HN
HN
O
CN
O
A
N
N
HN
Cl
O
H
H
B
B
A
H
HN
CN
B
A
N
H
H
EtO
A
N
B
H
49
61
O
35
neratinib
N
F
O
O
F
N
N
Ph =
R1
Cl
Cl
Cl
N
N
N
=
N
Figure 4. Design idea of the new compounds. (1) Open the B ring of lapatinib or neratinib to give the pyrimidine or 3-cyanopyridine compounds 16, 20, 29, 33 or 35. (2) Open
the A ring of lapatinib or neratinib to give the m-amino-N-phenylbenzamide compounds such as 49 and 61. (3) The similar substituents of lapatinib, neratinib or other typical
EGFR inhibitors are introduced to the related position on the new compounds.
O
OH
N
OH
N
Cl
N
N
Cl . HBr
O
I
I
CONH2
CN
d
CO2H
6
a, b, c
N
N
H
Br
a
H2N
Br
7
24
25
22
H
N
O
S
O
O
S
O
b
Br
NH2
CHO
O
.HCl
O
c
O
O
Br
N
N
8
O
N
HN
N
Cl
HN
N
Cl
H2
N
Cl
26
e
Cl
OH
R'
f
CN
OH
R
CN
g
I
e, f
O
O
NH2
S
d
F
N
N
N
OEt H2N
O
N
N
N
9
SH
27 R' = I
28 R' =
N
29a
29b
R =
R =
N
H
12
10
O
S
NH
O
O
O
O
F
O
F
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, reflux; (b) NH3ÁH2O, 79%; (c)
ICl, AcOH, 47%; (d) POCl3, diglyme, 100 °C, 86%; (e) CH3SO3H (cat.), EtOH, 70 °C, 89%;
(f) tributylethynylstannane, (Ph3P)2PdCl2, CuI, toluene, reflux, 79%; (g) 7 or 8,
NaOAc, (Ph3P)2PdCl2, NMP, 110 °C, 32–34%.
HN
N
Cl
HN
Cl
H2N
Cl
R
R'
13
g
i
N
N
N
O
was suspended in acetone (300 mL) and stirred for 20 min. The so-
lid was filtered and washed by acetone (50 mL Â 3). The combined
filtrate was concentrated to give the 6-methylpyrimidin-4-ol 10
(30 g, 58%) as a white solid. MS-ESI (m/z): 109.2(MÀH), 133.0
(M+Na); 1H NMR (CDCl3, d): 2.34(s, 3H), 6.32(s, 1H), 8.09(s, 1H).
Compound 10 (3.5 g, 0.0318 mol) was dissolved in a solution of
NaOH (1.5 g, 0.0375 mol) in H2O (50 mL). I2 (8.7 g, 0.0343 mol) was
added and the reaction solution was heated to reflux for 3 h. After
cooled to room temperature, the resulting solid was filtered and
washed by water, dried to give 5-iodo-6-methylpyrimidin-4-ol
11 (3.6 g, 49%) as a grey solid. 1H NMR (DMSO-d6, d): 2.45(s, 3H),
8.04(s, 1H).
POCl3 (5.3 mL, 0.057 mol) was added to the suspension of 11
(3.4 g, 0.0144 mol) in diglyme (60 mL) and the reaction mixture
was stirred at 90 °C for 2 h. After cooled to room temperature,
the reaction mixture was poured into 10% K2CO3 solution
(200 mL) and stirred for 1 h. The resulting solid was filtered and
washed by water, dried to give 4-chloro-5-iodo-6-methylpyrimi-
dine 12 (2.9 g, 79%).
14 R' = I
N
16a
16b
R =
R =
N
h
H
O
S
O
15 R' =
NH
O
Scheme 1. Reagents and conditions: (a) (COCl)2, THF, 0–28 °C, 6 h, 75%; (b) NaBH4,
MeOH, rt, 94%; (c) NaOMe, MeOH, reflux, 89%; (d) H2O2, 80 °C, 58%; (e) I2, NaOH,
H2O, reflux, 49%; (f) POCl3, diglyme, 90 °C, 79%; (g) CH3SO3H (cat.), EtOH, 70 °C, 94%;
(h) tributyl(vinyl)tin, (Ph3P)2PdCl2, CuI, toluene, 110 °C, 76%; (i) 7 or 8, NaOAc,
(Ph3P)2PdCl2, NMP, 110 °C, 45–47%.
(90 mL, 0.15 mol) was added and the resulting white solid was col-
lected by suction filtration, washed by water (150 mL Â 2), dried at
50 °C to give 2-mercapto-6-methylpyrimidin-4-ol 9 (66 g, 89%).
Compound 9 (66 g, 0.46 mol) was added portion-wise over 1 h
to a solution of 30% H2O2 (280 mL) and H2O (400 mL) at 70 °C
and stirred at 80–90 °C for another 1 h. After cooled down, Na2CO3
(ꢀ100 g) was added to the solution then stirred overnight to
quench the excess H2O2. Water was removed under reduced pres-
sure and the residual was dried at 50 °C to give a white solid, which

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3097
phyonsilicagel(eluent:PE/CH2Cl2 = 10/1–3/1)togiveN-(3-chloro-4-
((3-fluorobenzyl)oxy)phenyl)-6-methyl-5-vinylpyrimidin-4-amine
15 (0.24 g, 76%) as a light-yellow solid. MS-EI (m/z): 260, 277,
369(M+); 1H NMR (CDCl3, d): 2.40(s, 3H), 5.12(s, 2H), 5.63(d, 1H),
5.80(d, 1H), 6.61(dd, 1H), 6.81(s, 1H), 6.91(d, 1H), 7.01(t, 1H),
7.17–7.23(m, 2H), 7.31–7.39(m, 2H), 7.66(d, 1H), 8.51(br, 1H).
Compound 15 (40 mg, 0.108 mmol), 7 (45 mg, 0.16 mmol),
NaOAc (18 mg, 0.22 mmol) and (Ph3P)2PdCl2 (8 mg, 10 mol %)
were added into NMP (1.5 mL) under N2 atmosphere and heated
at 110 °C for 3 h. After cooled down to room temperature, the
solution was diluted with CH2Cl2 (40 mL). The organic layer was
washed with saturated NaHCO3 and brine, and dried over anhy-
drous Na2SO4. The solvent was removed under reduced pressure
to give the crude product, which was purified by column chroma-
tography on silica gel (eluent: CH2Cl2–CH2Cl2/MeOH/TEA = 100/5/
0.5) to give 16a (29 mg, 46%) as a light-yellow solid. MS-ESI (m/
z): 570.2(MÀH), 606.1(M+Cl), 572.2(M+H); 1H NMR (CDCl3, d):
2.35(s, 6H), 3.41(d, 2H), 5.05(s, 2H), 6.69(d, 1H), 6.77–6.90(m,
2H), 6.98(t, 1H), 7.10–7.21(m, 2H), 7.28–7.40(m, 1H), 7.65–
7.73(m, 2H), 8.03(s, 1H), 8.40(s, 1H).
O
S
R
N
NH
c
a, b
O
Z
14 or 27
R
N
O
Z
19a, 19b
32a, 32b Z = C-CN
Z = N
a, b
O
N
20a
Z = N
Z = N
R =
O
HN
Cl
N
R
N
.HBr
HO
Cl
17
O
I
N
Z
20b
12 or 25
R =
O
Cl
d
18b
Z = N
31b Z = C-CN
O
N
33a
33b
Z = C-CN R =
HN
Cl
O
N
R =
Z = C-CN
O
Cl
Scheme 3. Reagents and conditions: (a) trimethylsilylacetylene, (Ph3P)2PdCl2, CuI,
THF, TEA; (b) TBAF, THF, 56–85%; (c) 8, (Ph3P)2PdCl2, CuI, THF, TEA, 40 °C, 23–58%;
(d) K2CO3, DMF, 60 °C, 76–83%.
4.2.5. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methyl-5-
(2-(5-(((2-(methylsulfonyl)ethyl)amino)methyl)furan-2-
yl)vinyl)pyrimidin-4-amine (16b)
4.2.4. N-(3-(-2-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)-6-methylpyrimidin-5-yl)vinyl)phenyl)-4-(dimethylamino)
but-2-enamide (16a)
Compound12(1.6 g,6.28 mmol),commerciallyavailable3-chloro-
4-(3-fluorobenzyloxy)aniline 13 (1.6 g, 6.3 mmol) and CH3SO3H
Prepared in a similar manner as for 16a. Starting with 15 (80 mg,
0.216 mmol) and 8 (100 mg, 0.35 mmol) to produce 16b (55 mg,
45%) as a beige solid. MS-ESI (m/z): 569.0(MÀH), 604.9(M+Cl),
571.1(M+H), 593.1(M+Na); 1H NMR (CDCl3, d): 2.46(s, 3H), 2.94(s,
3H), 3.20(br, 4H), 3.88(s, 2H), 5.12(s, 2H), 6.28(d, 1H), 6.33(d, 1H),
6.88–6.95(m, 2H), 6.96–7.05(m, 3H), 7.18–7.22(m, 4H), 7.30–
7.35(m, 1H), 7.39(dd, 1H), 7.63(d, 1H), 8.48(s, 1H).
(20 lL, 5 mol %) were added into anhydrous EtOH (40 mL) and
the reaction mixture was heated at 70 °C for 2 h. After cooled down,
thealcoholsolutionwasdilutedwithwater(150 mL)andtheresulting
solidwasfiltered,washedwithwateranddriedtogiveN-(3-chloro-4-
((3-fluorobenzyl)oxy)phenyl)-5-iodo-6-methylpyrimidin-4-amine
14 (2.77 g, 94%) as a white solid. MS-ESI (m/z): 467.8(MÀH),
470.0(M+H); 1H NMR (CDCl3, d): 2.65(s, 3H), 5.15(s, 2H),
6.94(d, 1H), 7.02(t, 1H), 7.16(d, 1H), 7.22(d, 1H), 7.35(d, 2H), 7.67(s,
1H),8.39(s,1H).
Compound 14 (0.4 g, 0.85 mmol), tributyl(vinyl)tin (0.37 mL,
1.27 mmol), CuI (16 mg, 10 mol %) and (Ph3P)2PdCl2 (30 mg,
5 mol %) were suspended in toluene (12 mL) under N2 atmosphere
and heated at 110 °C for 4 h. After cooled down to room tempera-
ture, the solution was diluted with CH2Cl2 (60 mL). The organic
layer was washed with saturated NaHCO3 and brine, and dried over
anhydrousNa2SO4.Thesolventwasremovedunderreducedpressure
togivethecrudeproduct,whichwaspurifiedbycolumnchromatogra-
4.2.6. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methyl-5-
((5-(((2-(methylsulfonyl)ethyl)amino)methyl)furan-2-
yl)ethynyl)pyrimidin-4-amine (20a)
Compound 14 (0.21 g, 0.44 mmol), trimethylsilylacetylene
(85 lL, 0.6 mmol), CuI (8 mg, 10 mol %) and (Ph3P)2PdCl2 (15 mg,
5 mol %) were suspended in anhydrous THF (4 mL) under N2 atmo-
sphere and heated at 50 °C for 2 h. The solution was diluted with
CH2Cl2 (60 mL). The organic layer was washed twice with satu-
rated NaHCO3, and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure to give a brown flake, which
was dissolved into CH2Cl2 (4 mL) at 0 °C and treated dropwise with
a solution of TBAF (0.112 g, 0.44 mmol) in THF (2 mL). After stirred
at 0 °C for another 30 min, the reaction solution was diluted with
CH2Cl2 (60 mL). The organic layer was washed twice with brine,
O2N
d, e
O
O2N
O2N
O
CN
O2N
a, b
c
O
CN
COOH
CN
N
CO2Et
O
O
37
36
39
N
N
O
R
R2
O2N
HN
N
. 2 HCl
Cl
Cl
N
f
g
45
CN
35a - 35h
CN
i
40
43a, 43c, 43e, 43g
R = -NO2:
h
R = -NH2: 44a, 44c, 44e, 44g
Scheme 4. Reagents and conditions: (a) SOCl2, reflux; (b) ethyl cyanoacetate, NaOEt, 76%; (c) 95% DMSO/H2O, 110 °C, 81%; (d) DMF–DMA, toluene, 110 °C; (e) veratrylamine,
toluene, 110 °C, 85%; (f) POCl3, 110 °C, 71%; (g) 13, 26, 3-chloro-4-fluoroaniline (41) or 4-aminoacetophenone (42), DME, PyÁHCl, reflux, 75–90%; (h) Fe, NH4Cl, HCl, EtOH, 70–
80 °C, 80–90%; (i) 6, (COCl)2, THF, NMP, 0 °C to rt; or 45, pyridine, 0 °C to rt, 24–54%.

3098
Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
AcHN
EtO
COCl
NO2
AcHN
EtO
COOH
NO2
H2N
HO
COOH
HN
a, b, c
e
d
51
52
R2
R2
O
HN
g
AcHN
f
H2N
h
O
48a - 48f
49a - 49f
EtO
NO2
EtO
NO2
54a, 54c, 54e
53a, 53c, 53e
Scheme 5. Reagents and conditions: (a) Ac2O, AcOH, 60 °C, 92%; (b) C2H5Br, K2CO3, DMF, 60 °C, 96%; (c) fuming HNO3, AcOH, 25–35 °C, 85%; (d) SOCl2, reflux; (e) 13, 26 or 41,
CH2Cl2, 0 °C to rt; (f) K2CO3, THF–MeOH, rt, 33–69%; (g) 6, (COCl)2, THF, NMP, 0 °C to rt; or 45, pyridine, 0 °C to rt, 23–76%; (h) Zn, CaCl2, EtOH–H2O, reflux, 48–95%.
4.2.7. 3-Chloro-4-(pyridin-2-ylmethoxy)phenol hydrobromide
(17)
SO2Cl2 (8.3 mL, 0.103 mol) was added dropwise to a solution of
O2N
EtO
CO2H
O2N
EtO
COCl
a
4-methoxyphenol (12.4 g, 0.1 mol) in CHCl3 (70 mL) over 2 h and
stirred for another 20 h at room temperature. The volatiles were
removed to give 2-chloro-4-methoxyphenol as a light-yellow oil.
The above 2-chloro-4-methoxyphenol (16.0 g, 0.1 mol), 2-
(chloromethyl)pyridine hydrochloride (16.4 g, 0.1 mol), K2CO3
(27.6 g, 0.2 mol) and KI (0.83 g, 5 mol %) were suspended in DMF
(100 mL) and stirred at 60 °C for 12 h. The reaction suspension
was diluted with water (400 mL) and stirred, the resulting solid
was filtered, washed with water and dried to give 2-((2-chloro-4-
methoxyphenoxy)methyl)pyridine (21.5 g, 86%) as a grey solid.
1H NMR (CDCl3, d): 3.74(s, 3H), 5.20(s, 2H), 6.74(m, 1H), 6.89–
6.98(m, 2H), 7.20(m, 1H), 7.65–7.73(m, 2H), 8.59(s, 1H).
The above product (11 g, 0.044 mol) was added to 40% HBr
(90 mL) and the mixture was heated to reflux for 2 h. After cooled
to0 °C,theresultingsolidwasfiltered,washedbywater,driedtogive
17 (8.4 g, 61%) as an off-white solid. 1H NMR (CDCl3, d): 5.16(s, 2H),
6.61–6.92(m, 3H), 7.28–7.30(m, 1H), 7.68–7.79(m, 2H), 8.59(d, 1H).
55
56
R2
O
HN
R
d
b
c
50a - 50d
EtO
R = -NO2: 58a, 58c
59a, 59c
R = -NH2:
Scheme 6. Reagents and conditions: (a) SOCl2, reflux; (b) 26, CH2Cl2, 0 °C to rt; or 4-
(pyridin-3-yl) pyrimidin-2-amine (57), pyridine, 0 °C to rt, 51–82%; (c) Fe, HCl,
DME–MeOH, 60 °C, 84–98%; (d) 6, (COCl)2, THF, NMP, 0 °C to rt; or 45, pyridine, 0 °C
to rt, 19–49%.
R2
HN
O2N
COCl
R
a
b
c
O
61a - 61d
4.2.8. N-((5-((4-(3-Chloro-4-(pyridin-2-ylmethoxy)phenoxy)-6-
methylpyrimidin-5-yl)ethynyl)furan-2-yl)methyl)-2-
(methylsulfonyl)ethanamine (20b)
R = -NO :
62
64a, 64c, 64d
65a, 65c, 65d
2
R = -NH :
2
Compound 17 (0.8 g, 2.46 mmol) and K2CO3 (0.68 g, 4.9 mmol)
were suspended in DMF (8 mL) and stirred at room temperature
for 20 min. 12 (0.61 g, 2.4 mmol) and KI (18 mg, 5 mol %) were
added to the reaction mixture and stirred at 60 °C for 2 h. The reac-
tion solution was diluted with water (60 mL) and the resulting so-
lid was filtered, washed by water and dried to give 4-(3-chloro-4-
(pyridin-2-ylmethoxy)phenoxy)-5-iodo-6-methylpyrimidine 18b
Scheme 7. Reagents and conditions: (a) 13, CH2Cl2, 0 °C to rt; 57 or 4-(3-
fluorophenyl)pyrimidin-2-amine (63), pyridine, 0 °C to rt, 47–80%; (b) Fe, HCl,
DME–MeOH, 60 °C, 91–98%; (c) 6, (COCl)2, THF, NMP, 0 °C to rt; or 45, pyridine, 0 °C
to rt, 21–54%.
and dried over anhydrous Na2SO4. The solvent was removed under
reduced pressure to give the crude product, which was purified by
column chromatography on silica gel (eluent: PE/CH2Cl2 = 10/1–3/
1) to give N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-5-ethynyl-
6-methylpyrimidin-4-amine 19a (0.124 g, 77%). MS-ESI (m/z):
368.2(M+H); 1H NMR (CDCl3, d): 2.56(s, 3H), 3.86(s, 1H), 5.13(s,
2H), 6.93(d, 1H), 7.01(t, 1H), 7.01–7.25(m, 2H), 7.30–7.38(m, 1H),
7.39–7.47(m, 2H), 7.20(d, 1H), 8.54(s, 1H).
Compound 19a (35 mg, 0.095 mmol), 8 (40 mg, 0.14 mmol),
CuI (3 mg, 10 mol %) and (Ph3P)2PdCl2 (5 mg, 5 mol %)) were
suspended in anhydrous THF (2 mL) and Et3N (0.3 mL) under N2
atmosphere and heated at 45 °C for 12 h. The solvent was removed
under reduced pressure and the residual was diluted with CH2Cl2
(40 mL). The organic layer was washed with saturated NaHCO3
and brine, and dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure to give the crude product, which
was purified by column chromatography on silica gel (eluent: PE/
CH2Cl2 = 10/1–1/1) to give 20a (26 mg, 58%) as a beige solid. MS-
ESI (m/z): 566.8(MÀH), 602.9(M+Cl), 569.1(M+H); 1H NMR (CDCl3,
d): 2.56(s, 3H), 2.98(s, 3H), 3.18(s, 4H), 3.87(s, 2H), 5.14(s, 2H),
6.31(d, 1H), 6.69(d, 1H), 6.93(d, 1H), 7.01(dt, 1H), 7.18–7.24(m,
3H), 7.33(m, 1H), 7.42(dd, 1H), 7.72(m, 1H), 8.54(s, 1H).
(0.89 g, 76%) as
a beige solid. MS-ESI (m/z): 453.9(M+H),
475.9(M+Na); 1H NMR (DMSO-d6, d): 2.66(s, 3H), 5.31(s, 2H),
7.18(dd, 1H), 7.29(d, 1H), 7.38(dd, 1H), 7.44(d, 1H), 7.58(d, 1H),
7.89(dt, 1H), 8.45(s, 1H), 8.60(d, 1H).
In a similar manner as for 19a, started with 18b (0.5 g,
1.1 mmol) to produce 4-(3-chloro-4-(pyridin-2-ylmethoxy)phen-
oxy)-5-ethynyl-6-methylpyrimidine 19b (0.215 g, 56%) as a light-
yellow solid. 1H NMR (CDCl3, d): 2.68(s, 3H), 3.69(s, 1H), 5.28(s,
2H), 7.03(d, 2H), 7.27(t, 1H), 7.66(d, 1H), 7.77(dt, 1H), 8.56(s,
1H), 8.59(d, 1H).
In a similar manner as for 20a, started with 19b (70 mg,
0.2 mmol) and 8 (60 mg, 0.2 mmol) to produce 20b (42 mg, 38%)
as a beige solid. MS-ESI (m/z): 552.9(M+H); 1H NMR (CDCl3, d):
2.70(s, 3H), 2.99(s, 3H), 3.17(br, 4H), 3.85(s, 2H), 5.31(s, 2H),
6.28(d, 1H), 6.69(d, 1H), 7.04(d, 2H), 7.28(s, 1H), 7.52(dd, 1H),
7.65–7.80(m, 2H), 8.55(s, 1H), 8.60(br, 1H).
4.2.9. 4-Chloro-5-iodo-6-methylnicotinonitrile (25)
SOCl2 (5 mL, 0.066 mol) was added dropwise to a suspension of
4-hydroxy-6-methylnicotinic acid 22 (5 g, 0.033 mol) in anhydrous
MeOH (100 mL). The reaction solution was heated to reflux for 5 h.

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3099
The volatiles were removed to give a white solid, which was added
4.2.12. 4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-6-
to 28% NH3ÁH2O (50 mL) and the solution was stirred at room tem-
perature overnight. The reaction mixture was cooled in an ice bath
and the resulting solid was filtered, washed with water and dried
to give 4-hydroxy-6-methylnicotinamide 23 (4 g, 79%) as a white
solid. MS-ESI (m/z): 152.1(MÀH), 153.0(M+H); 1H NMR (DMSO-
d6, d): 2.24(s, 3H), 6.22(s, 1H), 7.37(br, 1H), 8.29(s, 1H), 9.58(br,
1H), 12.03(br, 1H).
ICl (1 mL, 20 mmol) was added dropwise over 20 min to a
solution of 23 (2 g, 13.1 mmol) in AcOH (20 mL) and the solu-
tion was stirred at room temperature overnight. The resulting
solid was filtered, washed with water and dried to give 4-
hydroxy-5-iodo-6-methylnicotinamide 24 (1.7 g, 47%) as a white
solid.
methyl-5-(2-(5-(((2-(methylsulfonyl)ethyl)amino)methyl)
furan-2-yl)vinyl)nicotinonitrile (29b)
In a similar manner as for 16a, started with 28 (60 mg,
0.16 mmol) and 8 (82 mg, 0.29 mmol) to produce 29b (31 mg,
34%) as a beige solid. MS-ESI (m/z): 576.0(MÀH), 611.8(M+Cl),
578.0(M+H); 1H NMR (CDCl3, d): 2.54(s, 3H), 2.88(s, 3H), 3.15(s,
4H), 3.83(s, 2H), 5.25(s, 2H), 6.27(d, 1H), 6.49(m, 1H), 6.68(m,
1H), 6.90–7.10(m, 2H), 7.52(dd, 2H), 7.60(d, 1H), 7.69–7.75(m,
3H), 8.35(s, 1H), 8.56(d, 1H).
4.2.13. 4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-6-
methyl-5-((5-(((2-(methylsulfonyl)ethyl)amino)methyl)furan-
2-yl)ethynyl)nicotinonitrile (33a)
In a similar manner as for 12, started with 24 (1.7 g, 6.1 mmol)
to produce 25 (1.5 g, 86%) as a faint-red solid. MS-ESI (m/z):
277.1(MÀH); 1H NMR (DMSO-d6, d): 2.81(s, 3H), 8.88(s, 1H).
In a similar manner as for 19a, started with 27 (0.5 g,
1.05 mmol) to produce 4-((3-chloro-4-(pyridin-2-ylmethoxy)
phenyl)amino)-5-ethynyl-6-methylnicotinonitrile 32a (0.335 g,
85%) as a faint-yellow solid. MS-EI (m/z): 282, 339, 374(M+); 1H
NMR (CDCl3, d): 2.69(s, 3H), 3.85(s, 1H), 5.42(s, 2H), 7.05(d, 1H),
7.15(m, 1H), 7.36(d, 1H), 7.75(d, 1H), 7.89(dt, 1H), 8.35(s, 1H),
8.61(d, 1H).
In a similar manner as for 20a, started with 32a (140 mg,
0.375 mmol) and 8 (110 mg, 0.375 mmol) to produce 33a (48 mg,
23%) as a brown solid. MS-ESI (m/z): 576.2(M+H), 598.1(M+Na);
1H NMR (CDCl3, d): 2.51(s, 3H), 2.89(s, 3H), 3.15(br, 4H), 3.87(s,
2H), 5.21(s, 2H), 6.35(d, 1H), 6.93(s, 1H), 7.01(dt, 1H), 7.14–
7.24(m, 3H), 7.31(m, 1H), 7.39(dd, 1H), 7.70(m, 1H), 8.58(s, 1H).
4.2.10. 3-Chloro-4-(pyridin-2-ylmethoxy)aniline (26)
2-(Chloromethyl)pyridine hydrochloride (16.4 g, 0.1 mol) and
K2CO3 (27.6 g, 0.2 mol) were suspended in DMF (100 mL) and stir-
red at room temperature for 30 min. 2-Chloro-4-nitrophenol
(17.4 g, 0.1 mol) and KI (0.83 g, 5 mol %) were added in and the
reaction mixture was stirred at 60 °C for 12 h. The reaction suspen-
sion was diluted with water (400 mL) and the resulting solid was
filtered, washed with water and dried to give 2-((2-chloro-4-nitro-
phenoxy)methyl)pyridine (26 g, 98%) as a white solid. Mp 149.2–
149.9 °C; MS-EI (m/z): 92, 229, 263(M+).
The above product (13.2 g, 0.05 mol), iron powder (11.2 g,
0.2 mol) and 12 M HCl (4 mL, 0.05 mol) were added into 90%
EtOH/H2O (200 mL) and the reaction mixture was stirred at
70 °C for 1 h. The dark solution was filtered through a Celite
pad. The filtrate was concentrated and the residual was dissolved
in CH2Cl2 (200 mL). The organic layer was washed twice with
water, and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to give 26 (10.9 g, 93%) as a
light-yellow solid. Mp 90.9–91.8 °C; MS-EI (m/z): 93, 142, 199,
234(M+); 1H NMR (DMSO-d6, d): 4.95(s, 2H), 5.07(s, 2H), 6.45(dd,
1H), 6.65(d, 1H), 6.90(d, 1H), 7.35(t, 1H), 7.55(d, 1H), 7.85(t, 1H),
8.55(d, 1H).
4.2.14. 4-(3-Chloro-4-(pyridin-2-ylmethoxy)phenoxy)-6-
methyl-5-((5-(((2-(methylsulfonyl)ethyl)amino)methyl)furan-
2-yl)ethynyl)nicotinonitrile (33b)
In a similar manner as for 18b, started with 25 (0.58 g,
2.1 mmol) and 17 (0.71 g, 2.2 mmol) to produce 4-(3-chloro-4-
(pyridin-2-ylmethoxy)phenoxy)-5-iodo-6-methylnicotinonitrile
31b (0.83 g, 83%) as a brown solid. MS-ESI (m/z): 478.0(M+H); 1H
NMR (DMSO-d6, d): 2.82(s, 3H), 5.27(s, 2H), 6.96(dd, 1H), 7.24(d,
1H), 7.32(d, 1H), 7.37(t, 1H), 7.57(d, 1H), 7.87(dt, 1H), 8.58(d,
1H), 8.83(s, 1H).
In a similar manner as for 19a, started with 31b (0.5 g,
1.05 mmol) to produce 4-(3-chloro-4-(pyridin-2-ylmethoxy)phen-
oxy)-5-ethynyl-6-methylnicotinonitrile 32b (0.32 g, 81%) as
a
4.2.11. N-(3-(2-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)
amino)-5-cyano-2-methylpyridin-3-yl)vinyl)phenyl)-4-
(dimethylamino)but-2-enamide (29a)
faint-yellow solid. 1H NMR (CDCl3, d): 2.75(s, 3H), 3.53(s, 1H),
5.26(s, 2H), 6.86(d, 1H), 6.94(s, 1H), 7.12(d, 1H), 7.25(t, 1H),
7.62(d, 1H), 7.76(dt, 1H), 8.57(d, 1H), 8.63(s, 1H).
In a similar manner as for 14, started with 25 (1.4 g, 5 mmol)
and 26 (1.3 g, 5.5 mmol) to produce 4-((3-chloro-4-(pyridin-2-
In a similar manner as for 20a, started with 32b (80 mg,
0.213 mmol) and 8 (62 mg, 0.213 mmol) to produce 33b (33 mg,
27%) as a brown solid. MS-ESI (m/z): 577.4(M+H), 599.3(M+Na);
1H NMR (DMSO-d6, d): 2.68(s, 3H), 2.99(s, 3H), 3.32(br, 4H),
3.77(s, 2H), 5.25(s, 2H), 6.41(s, 1H), 6.66(d, 1H), 7.15(d, 1H),
7.24(s, 1H), 7.30–7.39(m, 2H), 7.49–7.56(m, 2H), 7.83(d, 1H),
8.57(s, 1H), 8.89(s, 1H).
ylmethoxy)phenyl)amino)-5-iodo-6-methylnicotinonitrile
27
(2.1 g, 89%) as a off-white solid. 1H NMR (CD3OD, d): 2.82(s, 3H),
5.34(s, 2H), 7.22(s, 2H), 7.42(s, 1H), 7.46(d, 1H), 7.75(d, 1H),
7.95(t, 1H), 8.29(s, 1H), 8.61(d, 1H).
In a similar manner as for 15, started with 27 (0.6 g, 1.16 mmol)
to produce 4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-
6-methyl-5-vinylnicotinonitrile 28 (0.34 g, 79%) as a yellow solid.
MS-EI (m/z): 284, 341, 376(M+); 1H NMR (CDCl3, d): 2.48(s, 3H),
5.26(s, 2H), 5.53(d, 1H), 5.77(d, 1H), 6.44–6.56(m, 2H), 6.93–
7.03(m, 2H), 7.22(d, 1H), 7.62(d, 1H), 7.75(t, 1H), 8.36(s, 1H),
8.58(d, 1H).
4.2.15. 4-Chloro-5-(4-nitrophenyl)nicotinonitrile (40)
Ethyl cyanocaetate (24.3 mL, 0.227 mol) was added to a NaOEt
solution prepared from Na (4.76 g, 0.207 mol) and anhydrous EtOH
(200 mL) and the reaction solution was stirred at 50 °C for 1 h be-
fore cooled in an ice-water bath.
In a similar manner as for 16a, started with 28 (45 mg,
0.12 mmol) and 7 (50 mg, 0.176 mmol) to produce 29a (25 mg,
32%) as
(DMSO-d6, d): 2.48(s, 3H), 2.68(s, 6H), 3.49(d, 2H), 5.20(s, 2H),
6.63(d, 1H), 6.75–7.10(m, 3H), 7.21(d, 2H), 7.60(s, 1H), 7.72(t,
1H), 7.92(s, 1H), 8.31(s, 1H), 8.56(d, 1H), 9.76(br, 1H).
2-(4-Nitrophenyl)acetic acid (15 g, 0.083 mol) was mixed with
SOCl2 (150 mL) and heated to reflux for 1 h. The volatiles were re-
moved under reduced pressure to give a dark-brown oil, which was
dissolved into THF (100 mL) and added dropwise to the above
EtOH solution at 0 °C and stirred for another 1 h. The reaction mix-
ture was diluted with water (700 mL) and adjusted to pH ꢀ3 by
10% H2SO4. The resulting solid was filtered, washed with water
a
beige solid. MS-ESI (m/z): 579.2(M+H); 1H NMR

3100
Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
and dried to give ethyl 2-cyano-4-(4-nitrophenyl)-3-oxobutanoate
36 (18 g, 76%).
539.5(M+H), 561.4(M+Na). 1H NMR (CDCl3, d): 2.25(s, 6H),
3.08(d, 2H), 5.20(s, 2H), 6.19(d, 1H), 6.55(s, 1H), 6.86–6.98(m,
3H), 7.12(d, 1H), 7.20–7.25(m, 2H), 7.58(d, 1H), 7.65–7.76(m,
3H), 8.23(s, 1H), 8.33(s, 1H), 8.47(s, 1H), 8.55(d, 1H).
Compound 36 (18 g, 0.065 mol) was added to 95% DMSO/H2O
(100 mL). The resulting mixture was heated at 110 °C for 1 h. The
reaction mixture was diluted with water (500 mL) and adjusted
to pH ꢀ2 by 10% H2SO4. The resulting solid was filtered, washed
with water and dried to give 4-(4-nitrophenyl)-3-oxobutanenitrile
37 (10 g, 81%) as a tan solid. MS-ESI (m/z): 203.1(MÀH); 1H NMR
(DMSO-d6, d): 3.92(s, 2H), 4.01(s, 2H), 7.32(d, 2H), 8.05(d, 2H).
Compound 37 (6.1 g, 0.03 mol) and DMF–DMA (10 mL,
0.075 mol) were added to toluene (60 mL) and heated to reflux
for 6 h. Then veratrylamine (7 g, 0.036 mol) was added to the solu-
tion and the mixture was heated to reflux for another 2 h. After
cooled down to room temperature, the resulting solid was filtered,
washed with toluene and dried to give 1-(3,4-dimethoxybenzyl)-
4.2.17. N-(4-(4-((3-Chloro-4-(pyridin-2-
ylmethoxy)phenyl)amino)-5-cyanopyridin-3-yl)phenyl)-4-((4-
methylpiperazin-1-yl)methyl)benzamide (35b)
Commercially available 4-((4-methylpiperazin-1-yl)methyl)
benzoic acid dihydrochloride (5 g, 0.016 mol) was added into SOCl2
(40 mL)andthemixturewasheatedtorefluxfor24 h.Thevolatileswere
removed under reduced pressure to give 4-((4-methylpiperazin-1-
yl)methyl)benzoylchloridedihydrochloride(45)asawhitesolid.
Compound 45 (0.25 g, 0.767 mmol) and 44a (0.2 g,
0.467 mmol) were suspended in pyridine (4 mL) and stirred at
0 °C for 4 h. The reaction solution was diluted with water
(30 mL) and adjusted to pH ꢀ9 with 10% NaOH solution,
extracted with CH2Cl2. The organic layer was washed with water
and brine, and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to give the crude product,
which was purified by column chromatography on silica gel
(eluent: CH2Cl2–CH2Cl2/MeOH/TEA = 100/5/0.5) to give 35b
(0.164 g, 54%) as a faint-brown solid. MS-ESI (m/z): 644.6(M+H),
666.5(M+Na). 1H NMR (DMSO-d6, d): 2.21(s, 3H), 2.05(br, 8H),
3.53(s, 2H), 5.20(s, 2H), 7.02(d, 1H), 7.10(d, 1H), 7.20(d, 1H), 7.32–
7.44(m, 4H), 7.51(d, 1H), 7.70–7.92(m, 4H), 8.29(s, 1H), 8.48(s, 1H),
8.56(s, 2H), 10.36(s, 1H).
5-(4-nitrophenyl)-4-oxo-1,4-dihydropyridine-3-carbonitrile
39
(10 g, 85%) as
a
tan solid. MS-ESI (m/z): 390.2(MÀH),
392.2(M+H); 1H NMR (CDCl3, d): 3.87(s, 3H), 3.89(s, 3H), 5.04(s,
2H), 6.78(s, 1H), 6.83–6.93(m, 2H), 7.63(d, 1H), 7.02(d, 2H),
7.91(d, 1H), 8.00(s, 1H), 8.18(d, 2H).
Compound 39 (5 g, 0.0118 mol) and LiCl (4.2 g, 0.1 mol) were
added to POCl3 (50 mL) and the solution was heated to reflux for
3 h. The volatiles were removed under reduced pressure and the
residual was treated with water, adjusted pH ꢀ7 by 10% aqueous
NaOH. The resulting solid was filtered, washed with water and
dried to give 40 (2.2 g, 71%) as a tan solid. 1H NMR (DMSO-d6, d):
7.86(d, 2H), 8.39(d, 2H), 8.92(s, 1H), 9.19(s, 1H).
4.2.16. N-(4-(4-((3-Chloro-4-(pyridin-2-
ylmethoxy)phenyl)amino)-5-cyanopyridin-3-yl)phenyl)-4-
(dimethylamino)but-2-enamide (35a)
4.2.18. N-(4-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)-5-cyanopyridin-3-yl)phenyl)-4-(dimethylamino)but-2-
enamide (35c)
Compound 40 (0.66 g, 2.56 mmol), 26 (0.6 g, 2.56 mmol) and
pyridine hydrochloride (0.3 g, 2.56 mmol) were added to DME
(15 mL) and the solution was heated to reflux for 5 h. The reaction
mixture was diluted with water (100 mL) and the resulting solid
was filtered, washed with water and dried to give 4-((3-chloro-4-
(pyridin-2-ylmethoxy)phenyl)amino)-5-(4-nitrophenyl)nicotino-
nitrile 43a (0.88 g, 75%) as a faint-yellow solid. MS-ESI (m/z):
458.4(M+H), 480.3(M+Na); 1H NMR (DMSO-d6, d): 5.19(d, 2H),
6.99–7.08(m, 2H), 7.18(s, 1H), 7.37(m, 1H), 7.47(d, 1H), 7.67(d,
2H), 7.85(t, 1H), 8.21(d, 2H), 8.34(s, 1H), 8.57(d, 1H), 8.66(s, 1H),
8.72(s, 1H).
Compound 43a (0.86 g, 1.92 mmol), iron powder (0.65 g,
11.5 mmol), NH4Cl (0.1 g, 1.9 mmol) and 2 M HCl (2 mL) were added
into EtOH (20 mL) and the reaction solution were stirred at 70 °C for
1 h. The dark solution was filtered through a Celite pad. The filtrate
was concentrated to ꢀ10 mL and the residual was diluted with
water (20 mL). The resulting solid was filtered, washed with water
and dried to give 5-(4-aminophenyl)-4-((3-chloro-4-(pyridin-2-
ylmethoxy)phenyl)amino)nicotinonitrile 44a (0.77 g, 94%) as a tan
solid. MS-ESI (m/z): 428.2(M+H), 450.2(M+Na).
In a similar manner as for 43a, started with 40 (0.6 g,
2.31 mmol) and 13 (0.58 g, 2.31 mmol) to produce 4-((3-chloro-
4-((3-fluorobenzyl)oxy)phenyl)amino)-5-(4-nitrophenyl)nicotino-
nitrile 43c (0.89 g, 82%) as a grey solid. MS-ESI (m/z): 475.3(M+H);
1H NMR (DMSO-d6, d): 5.15(d, 2H), 7.04–7.06(m, 2H), 7.15–7.26(m,
4H), 7.45(m, 1H), 7.67(d, 2H), 8.23(d, 2H), 8.35(s, 1H), 8.67(s, 1H).
In a similar manner as for 44a, started with 43c (0.8 g,
1.68 mmol) to produce 5-(4-aminophenyl)-4-((3-chloro-4-((3-flu-
orobenzyl)oxy)phenyl)amino)nicotinonitrile 44c (0.67 g, 90%) as a
yellow solid. MS-ESI (m/z): 445.4(M+H).
In a similar manner as for 35a, started with 44c (0.2 g,
0.45 mmol) and 6 (0.15 g, 0.7 mmol) to produce 35c (63 mg, 25%)
as
a
white solid. MS-ESI (m/z): 554.4(MÀH), 590.5(M+Cl),
556.4(M+H); 1H NMR (CDCl3, d): 2.24(s, 6H), 3.07(d, 2H), 5.07(s,
2H), 6.20(d, 1H), 6.53(s, 1H), 6.83(d, 1H), 6.92–7.00(m, 2H),
7.12(d, 1H), 7.18(d, 2H), 7.27(m, 2H), 7.70(d, 2H), 8.23(s, 1H),
8.32(s, 1H), 8.47(s, 1H).
4.2.19. N-(4-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)
amino)-5-cyanopyridin-3-yl)phenyl)-4-((4-methylpiperazin-1-
yl)methyl)benzamide (35d)
Compound 6 (0.15 g, 0.7 mmol) and DMF (2
added into THF (4 mL) and the suspension was cooled in an ice-
water bath under N2 atmosphere. Oxalyl chloride (62 L,
lL, 5 mol %) were
In a similar manner as for 35b, started with 44c (0.2 g,
0.45 mmol) and 45 (0.25 g, 0.76 mmol) to produce 35d (116 mg,
l
0.7 mmol) was added and the reaction suspension was stirred at
26–30 °C for 1.5 h, then cooled again in ice-water bath. A solution
of 44a (0.2 g, 0.467 mmol) in NMP (3 mL) was added into the reac-
tion suspension and the mixture was stirred at 0 °C for 3 h. The
reaction solution was diluted with water (30 mL) and adjusted to
pH ꢀ9 with 10% NaOH solution, extracted with CH2Cl2. The organic
layer was washed with water and brine, and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure to give
the crude product, which was purified by column chromatography
on silica gel (eluent: CH2Cl2–CH2Cl2/MeOH/TEA = 100/5/0.5) to
give 35a (72 mg, 29%) as a faint-yellow solid. MS-ESI (m/z):
39%) as a
white solid. MS-ESI (m/z): 661.5(M+H); 1H NMR
(DMSO-d6, d): 2.19(s, 3H), 2.39(br, 8H), 3.53(s, 2H), 5.16(s, 2H),
7.03–7.28(m, 6H), 7.36–7.44(m, 5H), 7.83–7.91(m, 4H), 8.29(s,
1H), 8.47(s, 1H), 8.56(s, 1H), 10.32(s, 1H).
4.2.20. N-(4-(4-((3-Chloro-4-fluorophenyl)amino)-5-
cyanopyridin-3-yl)phenyl)-4-(dimethylamino)but-2-enamide
(35e)
In a similar manner as for 43a, started with 40 (0.8 g, 3.08 mmol)
and 3-chloro-4-fluoroaniline 41 (0.45 g, 3.08 mmol) to produce
4-((3-chloro-4-fluorophenyl)amino)-5-(4-nitrophenyl)nicotinonit-

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3101
rile 43e (0.95 g, 85%) as a faint-brown solid. MS-ESI (m/z):
369.2(M+H); 1H NMR (DMSO-d6, d): 7.06(s, 1H), 7.25(d, 2H),
7.68(m, 2H), 8.24(d, 2H), 8.44(s, 1H), 8.73(s, 1H), 8.91(s, 1H).
In a similar manner as for 44a, started with 43e (0.9 g,
2.4 mmol) to produce 5-(4-aminophenyl)-4-((3-chloro-4-fluoro-
phenyl)amino)nicotinonitrile 44e (0.73 g, 89%) as a yellow solid.
MS-ESI (m/z): 339.3(M+H).
Fuming HNO3 (120 mL, 3 mol) was added dropwise over 1 h to a
solution of 3-acetamido-4-ethoxybenzoic acid (220 g, 1 mol) in
AcOH(1.5 L)tokeeptheinnertemperaturebelow35 °C. Thereaction
solutionwasstirredat25–35 °Cforanother6 h.Thereactionsolution
was poured into water (4 L) and stirred, the resulting solid was fil-
tered, washed with water (500 mL Â 3) and dried at 50 °C to give 5-
acetamido-4-ethoxy-2-nitrobenzoic acid 51 (230 g, 85%) as a faint-
brown solid. Mp 234–236 °C (dec). ESI-MS (m/z) 267(MÀH). 1H
NMR (DMSO-d6, d): 1.40 (t, 3H, J = 6.9 Hz), 2.17 (s, 3H), 4.26 (q, 2H,
J = 6.9 Hz), 7.61 (s, 1H), 8.56 (s, 1H), 9.46 (s, 1H), 13.55 (br s, 1H).
13C NMR (DMSO-d6, d): 14.14, 24.12, 65.29, 107.35, 119.04, 120.87,
131.10, 144.23, 149.64, 165.65, 169.49. Anal. Calcd for C11H12N2O6:
C, 49.26; H, 4.51; N, 10.44. Found: C, 49.25; H, 4.58; N, 10.54.
Compound 51 (14 g, 0.052 mol) and SOCl2 (15 mL, 0.2 mol)
were suspended in CH2Cl2 (150 mL) and the reaction mixture
was heated to reflux for 1 h to give a clear solution. The volatiles
were removed under reduced pressure to give 52 as a white
solid.
In a similar manner as for 35a, started with 44e (0.2 g,
0.59 mmol) and 6 (0.19 g, 0.88 mmol) to produce 35e (78 mg,
29%) as
a
white solid. MS-ESI (m/z): 450.4(M+H); 1H NMR
(DMSO-d6, d): 2.17(s, 6H), 3.06(d, 2H), 6.28(d, 1H), 6.68–6.78(m,
1H), 7.01(m, 1H), 7.18–7.26(m, 2H), 7.33(d, 2H), 7.69(d, 2H),
8.37(s, 1H), 8.64(s, 1H), 8.69(s, 1H), 10.19(s, 1H).
4.2.21. N-(4-(4-((3-Chloro-4-fluorophenyl)amino)-5-
cyanopyridin-3-yl)phenyl)-4-((4-methylpiperazin-1-
yl)methyl)benzamide (35f)
In a similar manner as for 35b, started with 44e (0.2 g,
0.59 mmol) and 45 (0.29 g, 0.88 mmol) to produce 35f (140 mg,
43%) as a white solid. MS-ESI (m/z): 555.3(M+H); 1H NMR (CDCl3,
d): 2.29(s, 3H), 2.44(br, 8H), 3.54(s, 2H), 6.65(s, 1H), 6.95–7.12(m,
2H), 7.29(d, 2H), 7.41(d, 2H), 7.72(d, 2H), 7.81(d, 2H), 8.26(d,
1H), 8.41(s, 1H), 8.47(d, 1H).
4.2.25. N-(3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)-5-(4-
(dimethylamino)but-2-enamido)-4-ethoxy-2-nitrobenzamide
(48a) and 2-amino-N-(3-chloro-4-(pyridin-2-
ylmethoxy)phenyl)-5-(4-(dimethylamino)but-2-enamido)-4-
ethoxybenzamide (49a)
4.2.22. N-(4-(4-((4-Acetylphenyl)amino)-5-cyanopyridin-3-
yl)phenyl)-4-(dimethylamino)but-2-enamide (35g)
A solution of 26 (2.3 g, 0.01 mol) in CH2Cl2 (20 mL) was added
dropwise to the solution of 52 (2.9 g, 0.01 mol) in CH2Cl2 (30 mL)
at 0 °C and the reaction suspension was stirred for another
30 min. The resulting solid was filtered and dried to give a grey so-
lid, which was suspended with K2CO3 (1.38 g, 0.01 mol) into MeOH
(100 mL) and THF (40 mL) and stirred at room temperature for 3 h.
The reaction suspension was treated with 12 M HCl to pH ꢀ7. The
solvent were removed under reduced pressure and the residual
was added into water (40 mL) and stirred, the resulting solid was
filtered, washed with water, and dried to give 5-amino-N-(3-
chloro-4-(pyridin-2-ylmethoxy)phenyl)-4-ethoxy-2-nitrobenza-
mide 54a (3.1 g, 69%) at a bright-yellow solid.
In a similar manner as for 35a, started with 54a (0.44 g,
1 mmol) and 6 (0.42 g, 2 mmol) to produce 48a (135 mg, 25%) as
a light-yellow solid. MS-ESI (m/z): 552.1(MÀH), 587.8(M+Cl),
554.0(M+H); 1H NMR (CDCl3, d): 1.54(t, 3H), 2.28(s, 6H), 3.12(d,
2H), 4.24(q, 2H), 5.25(s, 2H), 6.18(d, 1H), 6.91–7.05(m, 2H),
7.22(t, 1H), 7.39(dd, 1H), 7.58(s, 1H), 7.62(d, 1H), 7.72(d, 1H),
7.78(d, 1H), 8.03(d, 2H), 8.57(d, 1H), 8.75(s, 1H).
In
a similar manner as for 43a, started with 40 (0.8 g,
3.08 mmol) and 4-aminoacetophenone 42 (0.42 g, 3.08 mmol) to
produce 4-((4-acetylphenyl)amino)-5-(4-nitrophenyl)nicotinonit-
rile 43g (0.96 g, 88%) as a faint-brown solid. MS-ESI (m/z):
359.2(M+H); 1H NMR (DMSO-d6, d): 2.44(s, 3H), 6.96(d, 2H),
7.65(d, 2H), 7.75(d, 2H), 7.96(d, 2H), 8.18–8.27(m, 5H), 8.55(s,
1H), 8.64(s, 1H), 8.90(s, 1H).
In a similar manner as for 44a, started with 43g (0.9 g,
2.5 mmol) to produce 4-((4-acetylphenyl)amino)-5-(4-aminophe-
nyl)nicotinonitrile 44g (0.74 g, 90%) as a yellow solid. MS-ESI (m/
z): 329.4(M+H).
In a similar manner as for 35a, started with 44g (0.2 g,
0.61 mmol) and 6 (0.19 g, 0.88 mmol) to produce 35g (68 mg,
24%) as a white solid. MS-ESI (m/z): 440.5(M+H), 462.5(M+Na);
1H NMR (DMSO-d6, d): 2.17(s, 6H), 2.45(s, 3H), 3.05(d, 2H),
6.91(d, 2H), 7.32(d, 2H), 7.66(d, 2H), 7.74(d, 2H), 8.60(s, 1H),
8.83(s, 1H), 9.11(br, 1H), 10.17(br, 1H).
Compound 48a (80 mg, 0.153 mmol), zinc powder (0.2 g,
3 mmol) and CaCl2 (85 mg, 0.76 mmol) were added into 80%
EtOH/H2O (10 mL) and the reaction mixture was stirred at reflux
for 10 h. The solvent was removed under reduced pressure and
the residual was dissolved in CH2Cl2 (30 mL). The organic layer
was washed twice with brine, and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure to give the
crude product, which was purified by column chromatography
4.2.23. N-(4-(4-((4-Acetylphenyl)amino)-5-cyanopyridin-3-
yl)phenyl)-4-((4-methylpiperazin-1-yl)methyl)benzamide (35h)
In a similar manner as for 35b, started with 44g (0.2 g,
0.61 mmol) and 45 (0.29 g, 0.88 mmol) to produce 35h (136 mg,
41%) as a white solid. MS-ESI (m/z): 545.5(M+H), 567.5(M+Na);
1H NMR (CDCl3, d): 2.25(s, 3H), 2.43(br, 8H), 2.48(s, 3H), 3.52(s,
2H), 6.97(d, 1H), 7.25(d, 1H), 7.38(d, 2H), 7.67(d, 1H), 7.81(d,
3H), 8.41(s, 1H), 8.48(s, 1H), 8.58(d, 1H).
on silica gel (eluent: CH2Cl2
– CH2Cl2/MeOH/TEA CH2Cl2–
CH2Cl2/MeOH/TEA = 100/5/0.5) to give 49a (55 mg, 48%) as a white
solid. MS-ESI (m/z): 524.1(M+H), 546.1(M+Na); 1H NMR (CDCl3, d):
1.45(t, 3H), 2.28(s, 6H), 3.01(d, 2H), 4.07(q, 2H), 5.25(s, 2H),
5.75(br, 2H), 6.12(d, 1H), 6.14(s, 1H), 6.88–6.97(m, 2H), 7.22(t,
1H), 7.35(dd, 1H), 7.62(s, 2H), 7.71–7.75(m, 2H), 8.39(s, 1H),
8.58(s, 2H)
4.2.24. 5-Acetamido-4-ethoxy-2-nitrobenzoyl chloride (52)
Ac2O (150 mL, 1.5 mol) was added dropwise over 1 h to a solu-
tion of 3-amino-4-hydroxybenzoic acid (155 g, 1 mol) in AcOH
(1 L) at 60 °C. The reaction solution was poured into water (3 L)
and stirred, the resulting solid was filtered, washed with water
(500 mL Â 3) and dried at 50 °C to give 3-acetamido-4-hydroxy-
benzoic acid (180 g, 92%) as a white solid, which was suspended
with K2CO3 (190 g, 1.4 mol) in DMF (1 L) and heated to 60 °C.
C2H5Br (105 mL, 1.4 mol) was added dropwise to the suspension
over 1 h. The reaction solution was poured into water (3 L) and
stirred, the resulting solid was filtered, washed with water
(500 mL Â 3) and dried at 50 °C to give 3-acetamido-4-ethoxyben-
zoic acid (200 g, 96%) as a white solid.
4.2.26. N-(3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)-4-ethoxy-
5-(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2-
nitrobenzamide (48b) and 2-amino-N-(3-chloro-4-(pyridin-2-
ylmethoxy)phenyl)-4-ethoxy-5-(4-((4-methylpiperazin-1-
yl)methyl)benzamido)benzamide (49b)
In a similar manner as for 35b, started with 54a (0.48 g,
1.1 mmol) and 45 (0.6 g, 1.35 mmol) to produce 48b (610 mg, 87%)

3102
Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
as
a
grey solid. MS-ESI (m/z): 657.2(MÀH), 692.8(M+Cl),
2H), 6.66(s, 1H), 7.40(t, 1H), 7.50–7.56(m, 2H), 7.96(d, 1H),
10.56(s, 1H).
In a similar manner as for 35a, started with 54e (0.24 g,
0.678 mmol) and 6 (0.21 g, 1 mmol) to produce 48e (110 mg,
659.2(M+H); 1H NMR (CDCl3, d): 1.57(t, 3H), 2.28(s, 3H), 2.48(br,
8H), 3.57(s, 2H), 4.27(q, 2H), 5.25(s, 2H), 6.93(d, 1H), 7.22(t, 1H),
7.41(dd, 1H), 7.49(d, 2H), 7.62(d, 2H), 7.72(d, 1H), 7.78(s, 1H),
7.80(d, 2H), 8.13(s, 1H), 8.57(d, 1H), 8.74(s, 1H), 8.83(s, 1H).
35%) as
a
light-yellow solid. MS-ESI (m/z): 465.4(M+H),
In a similar manner as for 49a, started with 48b (0.12 g,
0.18 mmol) to produce 49b (0.11 g, 95%) as a grey solid. MS-ESI
(m/z): 629.2(M+H); 1H NMR (CDCl3, d): 1.47(t, 3H), 2.32(s, 3H),
2.52(br, 8H), 3.56(s, 2H), 4.08(q, 2H), 5.24(s, 2H), 5.77(br, 2H),
6.17(s, 1H), 6.90(d, 1H), 7.21(t, 1H), 7.36(d, 1H), 7.43(d, 2H), 7.62(d,
2H), 7.72(s, 1H), 7.78(d, 2H), 8.30(d, 2H), 8.56(d, 1H), 8.66(s, 1H).
487.4(M+Na); 1H NMR (CDCl3, d): 1.52(t, 3H), 2.28(s, 6H), 3.12(d,
2H), 4.22(q, 2H), 6.15(d, 1H), 6.92–7.02(m, 1H), 7.08(t, 1H),
7.42(m, 1H), 7.54(s, 1H), 7.80(dd, 1H), 8.00(s, 1H), 8.60(s, 1H),
8.66(s, 1H).
In a similar manner as for 49a, started with 48e (66 mg,
0.142 mmol) to produce 49e (32 mg, 52%) as a grey solid. MS-ESI
(m/z): 435.4(M+H), 457.5(M+Na); 1H NMR (CDCl3, d): 1.45(t, 3H),
2.27(s, 6H), 3.12(d, 2H), 4.03(q, 2H), 5.75(br, 2H), 6.09(s, 1H),
6.86–6.98(m, 1H), 7.03(t, 1H), 7.42(m, 1H), 7.61(s, 1H), 7.77(dd,
1H), 8.44(s, 1H), 8.84(s, 1H).
4.2.27. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-5-(4-
(dimethylamino)but-2-enamido)-4-ethoxy-2-nitrobenzamide
(48c) and 2-amino-N-(3-chloro-4-((3-fluorobenzyl)oxy)
phenyl)-5-(4-(dimethylamino)but-2-enamido)-4-
ethoxybenzamide (49c)
4.2.30. N-(3-Chloro-4-fluorophenyl)-4-ethoxy-5-(4-((4-
methylpiperazin-1-yl)methyl)benzamido)-2-nitrobenzamide
(48f) and 2-amino-N-(3-chloro-4-fluorophenyl)-4-ethoxy-5-(4-
((4-methylpiperazin-1-yl)methyl)benzamido)benzamide (49f)
In a similar manner as for 35b, started with 54e (160 mg,
0.45 mmol) and 45 (220 mg, 0.68 mmol) to produce 48f (100 mg,
39%) as a yellow solid. MS-ESI (m/z): 570.4(M+H), 592.4(M+H);
1H NMR (CDCl3, d): 1.57(t, 3H), 2.30(s, 3H), 2.49(br, 8H), 3.58(s,
2H), 4.28(q, 2H), 7.10(t, 1H), 7.42–7.51(m, 3H), 7.61(s, 1H), 7.78–
7.81(m, 3H), 8.25(s, 1H), 8.73(s, 1H), 8.79(s, 1H).
In a similar manner as for 49a, started with 48f (50 mg,
0.088 mmol) to produce 49f (40 mg, 85%) as a tan solid. MS-ESI
(m/z): 540.5(M+H); 1H NMR (CDCl3, d): 1.48(t, 3H), 2.27(s, 3H),
2.45(br, 8H), 3.56(s, 2H), 4.07(q, 2H), 5.74(br, 2H), 6.14(s, 1H),
7.03(t, 1H), 7.40–7.48(m, 3H), 7.77–7.81(m, 3H), 8.29(s, 1H),
8.53(s, 1H), 8.86(s, 1H).
In a similar manner as for 54a, started with 52 (1 g, 3.7 mmol)
and 13 (0.94 g, 3.7 mmol) to give 5-amino-N-(3-chloro-4-((3-fluo-
robenzyl)oxy)phenyl)-4-ethoxy-2-nitrobenzamide 54c (0.59 g,
33%) as a tan solid. 1H NMR (DMSO-d6, d): 1.39(t, 3H), 4.17(q,
2H), 5.21(s, 2H), 6.51(br, 2H), 6.65(s, 1H), 7.15–7.31(m, 4H),
7.47(d, 2H), 7.55(s, 1H), 7.83(d, 1H), 10.38(s, 1H).
In a similar manner as for 35a, started with 54c (0.31 g,
0.674 mmol) and 6 (0.21 g, 1 mmol) to produce 48c (89 mg, 23%)
as a light-yellow solid. MS-ESI (m/z): 571.5(M+H), 593.5(M+Na);
1H NMR (CDCl3, d): 1.52(t, 3H), 2.27(s, 6H), 3.13(d, 2H), 4.22(q,
2H), 5.12(s, 2H), 6.17(d, 1H), 6.89(d, 1H), 6.95–7.04(m, 2H), 7.17–
7.24(m, 2H), 7.30–7.37(m, 1H), 7.43(dd, 1H), 7.56(s, 1H), 7.72(d,
1H), 8.02(s, 1H), 8.23(s, 1H), 8.67(s, 1H).
In a similar manner as for 49a, started with 48c (35 mg,
0.061 mmol) to produce 49c (29 mg, 87%) as a grey solid. MS-ESI
(m/z): 541.3(M+H), 563.6(M+Na); 1H NMR (CDCl3, d): 1.44(t, 3H),
2.29(s, 6H), 3.12(d, 2H), 4.04(q, 2H), 5.10(s, 2H), 6.12(s, 1H),
6.13(d, 1H), 6.22(br, 2H), 6.85–7.02(m, 3H), 7.17–7.22(m, 2H),
7.30–7.40(m, 2H), 7.62(s, 1H), 7.71(d, 1H), 8.50(s, 1H), 8.59(s, 1H).
4.2.31. N-(3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)-3-(4-
(dimethylamino)but-2-enamido)-4-ethoxybenzamide (50a)
4-Ethoxy-3-nitrobenzoic acid 55 (0.21 g, 1.1 mmol) was sus-
pended in SOCl2 (5 mL) and the reaction mixture was heated to re-
flux for 2 h to give a clear solution. The volatiles were removed
under reduced pressure to give 4-ethoxy-3-nitrobenzoyl chloride
56 as a light-yellow solid.
A solution of 26 (0.23 g, 1 mmol) in CH2Cl2 (3 mL) was added
dropwise to the solution of 56 (1.1 mmol) in CH2Cl2 (4 mL) at
0 °C and the reaction suspension was stirred for another 30 min.
The resulting solid was filtered and dried to give N-(3-chloro-4-
4.2.28. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-4-ethoxy-5-
(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2-
nitrobenzamide (48d) and 2-amino-N-(3-chloro-4-((3-
fluorobenzyl)oxy)phenyl)-4-ethoxy-5-(4-((4-methylpiperazin-
1-yl)methyl)benzamido)benzamide (49d)
In a similar manner as for 35b, started with 54c (120 mg,
0.182 mmol) and 45 (90 mg, 0.27 mmol) to produce 48d (230 mg,
76%) as a yellow solid. MS-ESI (m/z): 676.6(M+H); 1H NMR (CDCl3,
d): 1.54(t, 3H), 2.28(s, 3H), 2.49(br, 8H), 3.54(s, 2H), 4.23(q, 2H),
5.07(s, 2H), 6.86(d, 1H), 6.99(t, 1H), 7.16–7.21(m, 2H), 7.32(t,
1H), 7.40(d, 1H), 7.46(d, 2H), 7.53(s, 1H), 7.71–7.78(m, 3H),
8.41(s, 1H), 8.69(s, 1H), 8.75(s, 1H).
In a similar manner as for 49a, started with 48d (140 mg,
0.207 mmol) to produce 49d (110 mg, 82%) as a tan solid. MS-ESI
(m/z): 646.7(M+H); 1H NMR (CDCl3, d): 1.46(t, 3H), 2.28(s, 3H),
2.48(br, 8H), 3.56(s, 2H), 4.07(q, 2H), 5.08(s, 2H), 5.75(br, 2H),
6.16(s, 1H), 6.86(d, 1H), 6.98(t, 1H), 7.16–7.22(m, 2H), 7.30–
7.45(m, 4H), 7.74(d, 1H), 7.77(d, 2H), 8.31(s, 1H), 8.56(s, 1H),
8.67(s, 1H).
(pyridin-2-ylmethoxy)phenyl)-4-ethoxy-3-nitrobenzamide
58a
(0.35 g, 82%) as a yellow solid. 1H NMR (DMSO-d6, d): 1.37(t, 3H),
4.31(q, 2H), 5.26(s, 2H), 7.24(d, 1H), 7.36(dd, 1H), 7.50(d, 1H),
7.56(d, 1H), 7.62(dd, 1H), 7.87(dt, 1H), 7.95(d, 1H), 8.25(dd, 1H),
8.50(d, 1H), 8.59(d, 1H), 10.37(s, 1H).
Compound 58a (0.3 g, 0.71 mmol), iron powder (0.25 g,
4.2 mmol) and 2 M HCl (0.7 mL, 1.4 mmol) were added into DME
(40 mL) and MeOH (40 mL) and the reaction solution were stirred
at 60 °C for 5 h. The dark solution was filtered through a Celite pad.
The filtrate was concentrated under reduced pressure and the
residual was added into 5% K2CO3 solution and stirred to give 3-
amino-N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-4-ethoxy-
benzamide 59a (0.29 g, 98%) as a brown solid.
In a similar manner as for 35a, started with 59a (100 mg,
0.234 mmol) and 6 (100 mg, 0.467 mmol) to produce 50a (49 mg,
41%) as a white solid. MS-ESI (m/z): 509.1(M+H); 1H NMR (CDCl3,
d): 1.46(t, 3H), 2.27(s, 6H), 3.11(d, 2H), 4.14(q, 2H), 5.23(s, 2H),
6.17(d, 1H), 6.87–6.92(m, 2H), 7.20(m, 1H), 7.42(dd, 1H), 7.60(d,
1H), 7.71(m, 2H), 7.80(d, 1H), 7.89(s, 1H), 8.84(s, 1H), 8.56(d, 1H),
8.87(s, 1H).
4.2.29. N-(3-Chloro-4-fluorophenyl)-5-(4-(dimethylamino)but-
2-enamido)-4-ethoxy-2-nitrobenzamide (48e) and 2-amino-N-
(3-chloro-4-fluorophenyl)-5-(4-(dimethylamino)but-2-
enamido)-4-ethoxybenzamide (49e)
In a similar manner as for 54a, started with 52 (1 g, 3.7 mmol)
and 41 (0.54 g, 3.7 mmol) to give 5-amino-N-(3-chloro-4-fluoro-
phenyl)-4-ethoxy-2-nitrobenzamide 54e (0.45 g, 35%) as a yellow
solid. 1H NMR (DMSO-d6, d): 1.39(t, 3H), 4.17(q, 2H), 6.54(br,

Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
3103
4.2.32. N-(3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)-4-ethoxy-
3-(4-((4-methylpiperazin-1-yl)methyl)benzamido)benzamide
(50b)
Inasimilarmannerasfor35b,startedwith59a(120 mg,0.3 mmol)
and 45 (150 mg, 0.47 mmol) to produce 50b (90 mg, 49%) as an off-
white solid. MS-ESI (m/z): 612.1(MÀH), 647.9(M+Cl), 614.2[M+H];
1H NMR (CDCl3, d): 1.52(t, 3H), 2.30(s, 3H), 2.50(br, 8H), 3.58(s, 2H),
4.22(q, 2H), 5.27(s, 2H), 6.93(d, 1H), 7.00(d, 1H), 7.23(t, 1H), 7.45–
7.51(m, 3H), 7.63(d, 1H), 7.71–7.79(m, 2H), 7.83(d, 3H), 8.34(s, 1H),
8.58(d,1H),8.64(s,1H),8.96(d,1H).
54%) as a tan solid. MS-ESI (m/z): 599.2(MÀH), 635.0(M+Cl),
601.2(M+H); 1H NMR (DMSO-d6, d): 2.21(s, 3H), 2.33(s, 3H),
2.41(br, 8H), 3.54(s, 2H), 5.22(s, 2H), 7.17(dt, 1H), 7.21(s, 1H),
7.24(s, 1H), 7.26–7.33(m, 2H), 7.42–7.47(m, 3H), 7.65(dd, 1H),
7.88–7.92(m, 2H), 7.93–7.99(m, 1H), 10.25(d, 2H).
4.2.37. 5-(4-(Dimethylamino)but-2-enamido)-2-methyl-N-(4-
(pyridin-3-yl)pyrimidin-2-yl)benzamide (61c)
In a similar manner as for 58c, started with 2-methyl-5-nitro-
benzoyl chloride 62 (0.35 g, 2 mmol) and 57 (0.3 g, 1.74 mmol) to
produce
2-methyl-5-nitro-N-(4-(pyridin-3-yl)pyrimidin-2-
4.2.33. 3-(4-(Dimethylamino)but-2-enamido)-4-ethoxy-N-(4-
(pyridin-3-yl)pyrimidin-2-yl)benzamide (50c)
yl)benzamide 64c (0.31 g, 51%) as a yellow solid.
In a similar manner as for 59a, started with 64c (0.3 g,
0.895 mmol) to produce 5-amino-2-methyl-N-(4-(pyridin-3-
yl)pyrimidin-2-yl)benzamide 65c (0.26 g, 92%) as a brown solid.
In a similar manner as for 35a, started with 65c (230 mg,
0.753 mmol) and 6 (230 mg, 1.1 mmol) to produce 61c (64 mg,
21%) as a white solid. MS-ESI (m/z): 415.2(MÀH), 451.0(M+Cl),
417.1(M+H); 1H NMR (CDCl3, d): 2.20(s, 6H), 2.40(s, 3H), 3.05(d,
2H), 6.41(d, 1H), 6.90(m, 1H), 7.15(d, 1H), 7.38(dd, 1H), 7.43(d,
1H), 7.82(d, 1H), 7.97(s, 1H), 8.23(d, 1H), 8.62–8.68(m, 2H),
9.12(s, 1H), 9.70(s, 1H).
Commercially available 4-(pyridin-3-yl) pyrimidin-2-amine 57
(0.3 g, 1.74 mmol) was added to the solution of 56 (0.38 g, 2 mmol)
in pyridine (8 mL) at 0 °C and stirred at room temperature for 3 h.
The reaction solution was diluted with water (50 mL) and the
resulting solid was filtered, washed with water, and dried to give
4-ethoxy-3-nitro-N-(4-(pyridin-3-yl)pyrimidin-2-yl)benzamide
58c (0.31 g, 51%) as a light-brown solid. 1H NMR (DMSO-d6, d):
1.37(t, 3H), 4.32(q, 2H), 7.50(d, 1H), 7.75(dd, 1H), 7.99(d, 1H),
8.29(dd, 1H), 8.54(d, 1H), 8.74(d, 1H), 8.83(d, 1H), 8.88(d, 1H),
9.44(d, 1H), 11.33(br, 1H).
In a similar manner as for 59a, started with 58c (0.3 g,
0.89 mmol) to give 3-amino-4-ethoxy-N-(4-(pyridin-3-yl)pyrimi-
din-2-yl)benzamide 59c (0.23 g, 84%) as a tan solid.
In a similar manner as for 35a, started with 59c (220 mg,
0.65 mmol) and 6 (210 mg, 1 mmol) to produce 50c (65 mg, 22%)
as a white solid. MS-ESI (m/z): 447.3(M+H); 1H NMR (DMSO-d6,
d): 1.38(t, 3H), 2.77(s, 6H), 3.92(d, 2H), 4.11(q, 2H), 6.18(d, 1H),
6.70–6.81(m, 1H), 6.90(d, 1H), 7.34(s, 1H), 7.60(m, 1H), 7.89(d,
1H), 8.56(d, 1H), 8.74(d, 1H), 8.81(d, 1H), 9.39(s, 1H), 10.72(s, 1H).
4.2.38. 4-(3-Fluorophenyl)pyrimidin-2-amine (63)
1-(3-Fluorophenyl)ethanone (8 mL, 0.065 mol) and DMF–DMA
(25 mL, 0.2 mol) were heated to reflux in toluene (40 mL) for
24 h. Petroleum ether (30 mL) was added to the reaction solution
and let it cool to room temperature. The resulting solid was fil-
tered, washed with petroleum ether and dried to give 3-(dimethyl-
amino)-1-(3-fluorophenyl)prop-2-en-1-one (9.8 g, 78%).
The above product (5 g, 0.026 mol), guanidine nitrate (3.16 g,
0.026 mol) and NaOH (1.04 g, 0.026 mol) were added into i-PrOH
(40 mL) and heated to reflux for 2 h. After cooled in an ice-water
bath, the resulting solid was filtered, washed with i-PrOH and dried
to give 63 (3.1 g, 63%). 1H NMR (CDCl3, d): 5.18(br, 2H), 7.02(d, 1H),
7.17(dt, 1H), 7.39–7.47(m, 1H), 7.71–7.77(m, 2H), 8.37(d, 1H).
4.2.34. 4-Ethoxy-3-(4-((4-methylpiperazin-1-
yl)methyl)benzamido)-N-(4-(pyridin-3-yl)pyrimidin-2-
yl)benzamide (50d)
In a similar manner as for 35b, started with 59c (210 mg,
0.63 mmol) and 45 (310 mg, 1 mmol) to produce 50d (60 mg,
19%) as a tan solid. MS-ESI (m/z): 552.2(M+H); 1H NMR (CD3COCD3,
d): 1.51(t, 3H), 2.32(s, 3H), 2.55(br, 8H), 3.60(s, 2H), 4.31(q, 2H),
7.20(d, 1H), 7.50–7.55(m, 3H), 7.81(d, 1H), 7.89(d, 1H), 7.95(d,
2H), 8.59(d, 1H), 8.75(dd, 2H), 9.05(s, 1H), 9.40(s, 1H).
4.2.39. N-(4-(3-Fluorophenyl)pyrimidin-2-yl)-2-methyl-5-(4-
((4-methylpiperazin-1-yl)methyl)benzamido)benzamide (61d)
In a similar manner as for 58c, started with 62 (0.43 g,
2.3 mmol) and 63 (0.36 g, 1.9 mmol) to produce N-(4-(3-fluoro-
phenyl)pyrimidin-2-yl)-2-methyl-5-nitrobenzamide 64d (0.31 g,
47%) as a yellow solid.
4.2.35. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-5-(4-
(dimethylamino)but-2-enamido)-2-methylbenzamide (61a)
In a similar manner as for 58a, started with 2-methyl-5-nitro-
benzoic acid (0.2 g, 1.1 mmol) and 13 (0.25 g, 1 mmol) to produce
N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-2-methyl-5-nitroben-
zamide 64a (0.33 g, 80%) as a yellow solid.
In a similar manner as for 59a, started with 64a (0.3 g,
0.723 mmol) to produce 5-amino-N-(3-chloro-4-((3-fluoroben-
zyl)oxy)phenyl)-2-methylbenzamide 65a (0.28 g, 98%) as a brown
solid.
In a similar manner as for 59a, started with 64d (0.3 g,
0.88 mmol) to produce 5-amino-N-(4-(3-fluorophenyl)pyrimidin-
2-yl)-2-methylbenzamide 65d (0.25 g, 91%) as a brown solid.
In a similar manner as for 35b, started with 65d (160 mg,
0.47 mmol) and 45 (250 mg, 0.76 mmol) to produce 61d (95 mg,
39%) as a tan solid. MS-ESI (m/z): 539.2(M+H); 1H NMR (CDCl3,
d): 2.28(s, 3H), 2.44(br, 11H), 3.52(s, 2H), 5.29(s, 2H), 7.15(t, 1H),
7.22(d, 1H), 7.35–7.42(m, 3H), 7.63(d, 1H), 7.72(d, 2H), 7.84(d,
2H), 7.88(s, 1H), 8.44(s, 1H), 8.56(d, 1H), 9.10(br, 1H).
In a similar manner as for 35a, started with 65a (100 mg,
0.259 mmol) and 6 (82 mg, 0.39 mmol) to produce 61a (33 mg,
25%) as a white solid. MS-ESI (m/z): 494.0(MÀH), 529.9(M+Cl),
496.1(M+H); 1H NMR (CDCl3, d): 2.21(s, 6H), 2.35(s, 3H), 3.03(d,
2H), 5.09(s, 2H), 6.17(dd, 1H), 6.82–6.91(m, 2H), 6.95–7.07(m,
1H), 7.15–7.23(m, 2H), 7.32(d, 1H), 7.47(t, 2H), 7.67(d, 1H),
7.81(s, 1H), 8.30(s, 1H), 8.93(s, 1H), 8.98(s, 1H).
4.3. Biology
4.3.1. EGFR and ErbB-2 kinase assays by DELFIA/time-resolved
fluorometry
Compounds were dissolved in DMSO and diluted to the appro-
priate concentrations with 25 mM HEPES at pH 7.4. In each well,
10
12.5 ng for ErbB-2) of recombinant enzyme (1:80 dilution in
100 mM HEPES) for 10 min at room temperature. Then 10 L of
5Â buffer (containing 20 mM HEPES, 2 mM MnCl2, 100
Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM ATP–50 mM MgCl2
lL of compound was incubated with 10 lL (5 ng for EGFR or
4.2.36. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-2-methyl-5-
(4-((4-methylpiperazin-1-yl)methyl)benzamido)benzamide
(61b)
l
lM
l
In a similar manner as for 35b, started with 65a (110 mg,
0.286 mmol) and 45 (140 mg, 0.43 mmol) to produce 61b (93 mg,
was added for 1 h. Positive and negative controls were included
in each plate by incubation of enzyme with or without ATP–MgCl2.

3104
Y. Mao et al. / Bioorg. Med. Chem. 21 (2013) 3090–3104
P.; Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards,
L. Bioorg. Med. Chem. Lett. 2001, 11, 1191.
6. Rusnak, D. W.; Lackey, K. E.; Wood, E. R.; Alligood, K. J.; Rhodes, N.; Knight, W.
B.; Gilmer, T. M. Mol. Cancer Ther. 2001, 1, 85.
7. Normanno, N.; Bianco, C.; Strizzi, L.; Mancino, M.; Maiello, M. R.; DeLuca, A.;
Caponigro, F.; Salomon, D. S. Curr. Drug Targets 2005, 6, 243.
8. (a) Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya,
N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X. Q.; Rabindran, S. K.; Gruber, B.
C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y. F.; Greenberger, L. M.;
Tsou, H. R. J. Med. Chem. 2003, 46, 49; (b) Tsou, H. R.; Overbeek-Klumpers, E. G.;
Hallett, W. A.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Michalak, R. S.; Shen, R.;
Shi, X.; Wang, Y. F.; Upeslacis, J.; Wissner, A. J. Med. Chem. 2005, 48, 1107.
9. Traxeler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett, H.; Buchdunger, E.;
Meyer, T.; Mueller, M.; Furet, P. J. J. Med. Chem. 1997, 40, 3601.
At the end of incubation, liquid was aspirated, and plates were
washed three times with wash buffer. A 75 L (400 ng) sample of
l
europiumlabeled anti-phosphotyrosine antibody was added to
each well for another 1 h of incubation. After washing, enhance-
ment solution was added and the signal was detected with excita-
tion at 340 nm and emission at 615 nm. The IC50 was obtained
from curves of percentage inhibition.
4.3.2. Cell proliferation assay
The cell proliferation assays were done with two human carci-
noma cell lines: A-549 and HL60. Cells were plated in 96-well
plates at densities of 5 Â 104/mL in RPMI-1640 medium supple-
mented with 5% fetal bovine serum. On the next day, compounds
were dosed at 0.5, 5, 50, 500, and 5000 ng/mL concentrations and
the cells were cultured for 2 days. At the end of incubation, cell
survival was determined by the sulforhodamine B assay. The IC50
values were obtained from the growth curves.
10. Palmer, B. D.; Trumpp-Kallmeyer, S.; Fry, D. W.; Nelson, J. M.; Showalter, H. D.
H.; Denny, W. A. J. Med. Chem. 1997, 40, 1519.
11. (a) Cha, M. Y.; Lee, K. O.; Kang, S. J.; Jung, Y. H.; Song, J. Y.; Choi, K. J.; Byun, J. Y.;
Lee, H. J.; Lee, G. S.; Park, S. B.; Kim, M. S. J. Med. Chem. 2012, 55, 2846; (b)
Suzuki, N.; Shiota, T.; Watanabe, F.; Haga, N.; Murashi, T.; Ohara, T.; Matsuo, K.;
Omori, N.; Yari, H.; Dohi, K.; Inoue, M.; Iguchi, M.; Sentou, J.; Wada, T. Bioorg.
Med. Chem. Lett. 2012, 22, 456.
12. (a) Waterson, A. G.; Stevens, K. L.; Reno, M. J.; Zhang, Y. M.; Boros, E. E.; Bouvier,
F.; Rastagar, A.; Uehling, D. E.; Dickerson, S. H.; Reep, B.; McDonald, O. B.;
Wood, E. R.; Rusnak, D. W.; Alligood, K. J.; Rudolph, S. K. Bioorg. Med. Chem. Lett.
2006, 16, 2419; (b) Suzuki, N.; Shiota, T.; Watanabe, F.; Haga, N.; Murashi, T.;
Ohara, T.; Matsuo, K.; Oomori, N.; Yari, H.; Dohi, K.; Inoue, M.; Iguchi, M.;
Sentou, J.; Wada, T. Bioorg. Med. Chem. Lett. 2011, 21, 1601.
Acknowledgment
This work was supported by grants from the National High
Technology Research and Development Program of China
(2012AA020302).
13. Wong, K. K. Clin. Cancer Res. 2007, 13, 4593.
14. (a) Tsou, H. R.; Overbeek-Klumpers, E. G.; Hallett, W. A.; Reich, M. F.; Floyd, M.
B.; Johnson, B. D.; Michalak, R. S.; Nilakantan, R.; Discafani, C.; Golas, J.;
Rabindran, S. K.; Shen, R.; Shi, X.; Wang, Y. F.; Upeslacis, J.; Wissner, A. J. Med.
Chem. 2005, 48, 1107; (b) Petrov, K. G.; Zhang, Y. M.; Carter, M.; Cockerill, G. S.;
Dickerson, S.; Gauthier, C. A.; Guo, Y.; Mook, R. A.; Rusnak, D. W.; Walker, A. L.;
Wood, E. R.; Lackey, K. E. Bioorg. Med. Chem. Lett. 2006, 16, 4686.
Supplementary data
15. (a) Liu, Z. D.; Li, D. W.; Li, S. K.; Bai, D. L.; He, X. C.; Hu, Y. H. Tetrahedron 1931,
2007, 63; (b) Foster, H. M.; Snyder, H. R. Org. Synth. 1963, 4, 638; (c) Wissner,
A.; Rabindran, S. K.; Tsou, H. R. PCT Int. Appl. WO 2005034955, 2005.
16. (a) Scott, W. J.; Crisp, G. T.; Stille, J. K. Org. Synth. 1993, 8, 97; (b) Casado, A. L.;
Espinet, P. J. Am. Chem. Soc. 1998, 120, 8978.
17. Beletskaya, I. P.; Cheprakov, A. V. Chem. Rev. 2000, 100, 3009.
18. (a) Dushin, R. G.; Nittoli, T.; Ingalls, C.; Boschelli, D. H.; Cole, D. C.; Wissner, A.;
Lee, J.; Yang, X.; Morgan, P.; Brennan, A.; Chaudhary, D. Bioorg. Med. Chem. Lett.
2009, 19, 2461; (b) Cole, D. C.; Asselin, M.; Brennan, A.; Czerwinski, R.;
Ellingboe, J. W.; Fitz, L.; Greco, R.; Huang, X.; Joseph-McCarthy, D.; Kelly, M. F.;
Kirisits, M.; Lee, J.; Li, Y.; Morgan, P.; Stock, J. R.; Tsao, D. H.; Wissner, A.; Yang,
X.; Chaudhary, D. J. Med. Chem. 2008, 51, 5958.
19. (a) Mao, Y. J.; Li, J. F.; Zheng, J., Liu, Z.; Xie, K.; Li, H. H.; Shi, J.; Li, Y.; Shen, J. S.
PCT Int. Appl. WO 2010045785, 2010.; (b) Zhang, Q.; Mao, Y. J.; Liu, Z.; Xie, K.;
Zhu, Y.; Wei, Y. B.; Jiang, X. R.; Shen, J. S. Heterocycles 2011, 83, 2851; (c) Mao, Y.
J.; Liu, Z.; Yang, X. J.; Xia, X. F.; Zhang, R. X.; Li, J. F.; Jiang, X. R.; Xie, K.; Zheng, J.;
Zhang, H.; Suo, J.; Shen, J. S. Org. Process Res. Dev. 1970, 2012, 16.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.053.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
1. Supuran, C. T.; Scozzafava, A. Expert Opin. Ther. Patents 2004, 14, 35.
2. (a) Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005, 5, 341; (b) Nahta, R.; Shabaya,
S.; Ozbay, T.; Rowe, D. L. Curr. Pharm. Pers. Med. 2009, 7, 263.
3. Olayioye, M. A.; Neve, R. M.; Lane, H. A.; Hynes, N. E. EMBO J. 2000, 19, 3159.
4. Kamath, S.; Buolamwini, J. K. Med. Res. Rev. 2006, 26, 569.
5. (a) Khalil, M. Y.; Grandis, J. R.; Shin, D. M. Exp. Rev. Anticancer Ther. 2003, 3, 367;
(b) Baker, A. J.; Gibson, K. H.; Grudy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M.