Discovery of novel 2-aminopyridine-3-carboxamides as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.07.007

A series of 2-aminopyridine-3-carboxamide derivatives against c-Met were designed and synthesized by employing bioisosteric replacement of heterocyclic moieties with the amide bond. The structure-activity relationship (SAR) at various positions of the sca

Full text of DOI:10.1016/j.bmc.2012.07.007

Bioorganic & Medicinal Chemistry 20 (2012) 5169–5180
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel 2-aminopyridine-3-carboxamides as c-Met kinase inhibitors
Dengyou Zhang a, , Jing Ai b, , Zhongjie Liang a, , Chunpu Li a, Xia Peng b, YinChun Ji b, Hualiang Jiang a,c
,
a,
a,
Meiyu Geng b, , Cheng Luo , Hong Liu
⇑
⇑
⇑
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Shanghai 201203, PR China
b Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
c School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR 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 2-aminopyridine-3-carboxamide derivatives against c-Met were designed and synthesized by
employing bioisosteric replacement of heterocyclic moieties with the amide bond. The structure–activity
relationship (SAR) at various positions of the scaffold was explored. In this study, a promising compound
Received 13 May 2012
Revised 3 July 2012
Accepted 4 July 2012
Available online 16 July 2012
(S)-24o with a c-Met IC50 of 0.022 lM was identified. The compound exhibited dose-dependent inhibi-
tion of the phosphorylation of c-Met as well as downstream signaling in EBC-1 cells. Furthermore, the
interactive binding model of (S)-24o with c-Met was elucidated by virtue of a molecular modeling study.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Receptor tyrosine kinase
c-Met
2-Aminopyridine-3-carboxamide
1. Introduction
tures feature an aminopyridine core responsible for H-bonding
with the hinge region, an aryl fragment (P1 part) extending into
c-Met is a receptor tyrosine kinase (RTK), consisting of a disul-
fide linked -chain and b-chain. c-Met is known to be a pleiotropic
the inside pocket and a substituent (P2 part) facing to the solvent
accessible region. Crizotinib (Fig. 1A, 1) developed by Pfizer scien-
a
cytokine owing to its regulation of widespread effectors. Activation
of c-Met occurs upon binding with hepatocyte growth factor (HGF)
and interaction with other membrane receptors.1 Deregulation of
c-Met, frequently associated with Met gene amplification, c-Met
overexpression or mutations, has been documented in most solid
tumors and hematological malignancies.2,3 Furthermore, c-Met
signaling might be responsible for the emergence of the resistance
mechanism in chemotherapy, radiotherapy and other RTK based
target therapy.4 Therefore, c-Met kinase is a promising therapeutic
target for cancer.
To date, the main strategies employed to interrupt the c-Met
signaling pathway involve blocking the interaction between c-
Met and HGF via antibodies5–16 or antagonists17–21 and interfering
with the active site of the kinase domain with small -molecule ki-
nase inhibitors.22–41 Recently, among the various strategies, the
use of small molecules modulating c-Met has captured extensive
attention, resulting in a respectable number of compounds reach-
ing the clinical evaluation.42
tists displayed the c-Met inhibition with a Ki value of 0.002 l
M.26
The co-crystal structure of crizotinib (Fig. 1B) bound to c-Met re-
vealed that the 2,6-dichloro-3-fluorobenzyloxy fragment was able
to adopt an ideal conformation for involvement in a
p–p interac-
tion with Y1230, indicating that an aryl fragment linked with a
flexible linker at the C-3 position of the aminopyridine core was
tolerated. Lee et al.28 reported a series of aminopyridines substi-
tuted with benzoxazole, among which compound 4 showed highly
potent c-Met inhibition (IC50 = 0.003 lM). Besides, aminopyridine
analogs substituted with benzimidazole (e.g., compound 5)45,46
or tetrazole43 linked with the aryl fragments (e.g., compound 3)
have also been presented. Hence, a rigid substituent or linker
was compatible as well at the C-3 position of the aminopyridine
core.
Bioisosteric replacement is a fundamental tactical approach
widely used in medicinal chemistry.47 Replacement of heterocyclic
moieties with the amide bond is a common means deployed in
analogs design. Since the aminopyridine analogs with the imidaz-
ole (5), oxazole (4) or tetrazole (3) at the C-3 position displayed po-
tency against c-Met, we surmised the replacement the imidazole,
oxazole or tetrazole with the amide bond might give rise to novel
c-Met kinase inhibitors (Fig. 2). Meanwhile, according to the co-
crystal structural information (Fig. 1B), the introduction of suitable
2-Aminopyridine based c-Met inhibitors (Fig. 1A) have been
widely explored in recent years.26,28,43–46 Generally, these struc-
⇑
Corresponding authors. Tel.: +86 21 50806600; fax: +86 21 50807088.
E-mail addresses: mygeng@mail.shcnc.ac.cn (M. Geng), cluo@mail.shcnc.ac.cn
(C. Luo), hliu@mail.shcnc.ac.cn (H. Liu).
aryl fragments on the amide bond, engaging in a
p–p interaction
with Y1230, would be appropriate. Herein, we disclose our efforts
These authors contributed equally to this study.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.007

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D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
F
1B
1A
P 1
Cl
F
P 1
F
P 1
Cl
O
N
F
O
Cl
N
N
N
N
N
N
O
N
N
N
N
Cl
HN
O
HN
N
NH2
P 2
N
NH2
HN
NH2
P 2
N
3
P 2
2
1
N
N
P 1
P 1
N
O
HN
O
N
N
N
N
NH2
P 2
N
NH2
P 2
N
5
4
Figure 1. A. Selected examples of amino-pyridine based c-Met kinase inhibitors. B. Cocyrstal structure of crizotinib (1) bound to c-Met (2-WGJ).
O
P 1
NH2
R
NH
NHBoc
R
NBoc
R
Br
iv
R
OH
NH2
ii
i
iii
+
R
N
N
N
N
N
N
8
Aryl
P 2
14j-14l
11
12j-12l
13j-13l
P 1
R1
R2
O
NH2
N
N
bioisostere
R
3
Aryl
P 2
R
P 1
N
NH2
N
X
N
6
v
Br
N
O
Aryl
P 2
N
O
N
NH2
N
NH2
N
NH2
16j-16l
15j-15l
X = NH, O
4, 5
Scheme 2. Reagents and conditions: (i) (Boc)2O, THF, rt; (ii) NaH, MeI, DMF, 0 °C-rt;
(iii) CH2Cl2/TFA (4:1), rt; (iv) HATU, DIPEA, DMF, 0 °C-rt; (v) 1-methyl-4-(4,4,5,5-
tetramethyl-1,3, 2-dioxaborolan-2-yl)-1H-pyrazole, Pd(PPh3)2Cl2, Na2CO3, DME/
H2O (4:1), 80 °C.
Figure 2. Design of novel 2-aminopyridine-3-carboxamides based on known c-Met
kinase inhibitors.
to discover novel 2-aminopyridine-3-carboxamides as c-Met
inhibitors.
1H-pyrazole under Suzuki coupling conditions furnished the desired
products (16j–16l).
The synthesis of compounds (24m, 24o–24s) was described in
Scheme 3. Reduction of 2,6-dichloro-3-fluoroacetophenone with
NaBH4 afforded the benzyl alcohol 18, which was converted into
the mesylate 19. Substitution of 19 with sodium azide, followed
by reduction of the resulting benzyl azide using zinc and ammo-
nium chloride (Zn/NH4Cl), delivered intermediate 21. Compounds
24m, 24o–24s, 25 were similarly obtained as shown in Scheme 1.
The removal of Boc group of 25 gave compound 24n. Compounds
29 and 30 were prepared according to Scheme 4. 2-Amino-5-bro-
monicotinic acid 8 was smoothly converted into the intermediate
26 in the presence of HCONH2 under microwave irradiation. Alkyl-
ation of 26, followed by Suzuki-coupling, furnished compound 29.
Treatment of 29 with trifluoroacetic acid (TFA) in dichloromethane
(CH2Cl2) afforded compound 30.
2. Chemistry
Our synthesis commenced with bromination of 2-aminonicotinic
acid, followed by coupling with various commercial available ben-
zylamines, to give the intermediates 9a–9i as shown in Scheme 1.
Suzuki coupling of 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)-1H-pyrazole with 9 smoothly afforded compounds
10a–10i. The preparation of compounds (16j–16l) was outlined in
Scheme 2. Protection of benzylamines 11, followed by sequential
methylation and deprotection, provided N-methyl benzylamines
14, which were condensed with 8 to give 2-amino-N-benzyl-5-bro-
mo-N-methylpyridine-3-carboxamides 15j–l. Treatment of 15j–l
with
1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
R
R
O
O
HN
N
Br
HN
OH
NH2
i
ii
iii
OH
NH2
Br
N
O
O
N
N
N
NH2
9a-9i
7
N
NH2
8
10a-10i
Scheme 1. Reagents and conditions: (i) Br2, HAc, rt; (ii) benzylamines, HATU, DIPEA, DMF, 0 °C-rt; (iii) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole, Pd(PPh3)2Cl2, Na2CO3, DME/H2O (4:1), 80 °C.

D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
5171
O
HO
MsO
Cl
N3
Cl
F
Cl
ii
i
iii
Cl
Cl
Cl
Cl
F
Cl
Cl
F
F
17
18
19
20
21
iv
F
F
H2N
Cl
Cl
HN
Cl
Cl
Cl
v
vi
O
+
HN
Aryl
B
Aryl
O
F
Br
O
O
23
N
NH2
N
NH2
24m, 24o-24s
22
F
F
vi
Cl
Cl
vii
Cl
Cl
N
N
HN
N
HN
N
BocN
HN
O
O
N
25
NH2
N
NH2
24n
Scheme 3. Reagents and conditions: (i) NaBH4, MeOH, rt; (ii) MsCl, Et3N, 0 °C-rt; (iii) NaN3, DMF, 50 °C; (iv) Zn, NH4Cl, EtOH/H2O (3:1), rt; (v) 8, HATU, DIPEA, DMF, 0 °C-rt;
(vi) Pd(PPh3)4, KFꢁ2H2O, DME/H2O/EtOH (7:3:2), 110 °C, Mw; (vii) CH2Cl2/TFA (4:1), rt.
NBoc
O
O
N
O
N
Cl
F
N
N
O
ii
Br
Br
i
Br
OH
NH
N
+
N
NH2
B
N
N
Cl
27
O
26
8
iii
O
28
N
N
N
O
N
Cl
F
Cl
iv
N
HN
BocN
N
N
N
Cl
N
N
29
Cl
F
30
Scheme 4. Reagents and conditions: (i) HCONH2, Mw, 150 °C; (ii) 19, NaH, DMF, 0–80 °C; (iii) Pd(PPh3)4, KF.2H2O, DME/H2O/EtOH (7:3:2), 110 °C, Mw; (iv) CH2Cl2/TFA (4:1),
rt.
H2SO4, and the plate was read using a multiwell spectrophotome-
ter (VERSAmax™, Molecular Devices, Sunnyvale, CA,USA) at
490 nm. The inhibition rate (%) was calculated using the following
equation: [1-(A490/A490 control)] ꢀ 100%. IC50 values were calcu-
lated from the inhibition curves.
3. Biology
3.1. ELISA kinase assay
The tyrosine kinase activities were evaluated using enzyme-
linked-immunosorbent assay (ELISA). Briefly, 96-well plates were
3.2. Cell proliferation assay
precoated with 20
lg/ml Poly (Glu,Tyr) 4:1 (Sigma). 50 lL of
10 M 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 each well. Various concentrations of com-
pounds diluted in 10 lL of 1% DMSO (v/v) were added to each reac-
tion 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 of 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. A solution
l
Cells were seeded in 96-well tissue culture plates. The following
day, cells were exposed to compounds at various concentrations
and further cultured for 72 h. Finally, cell proliferation was deter-
mined using sulforhodamine B (SRB; Sigma) assay. Briefly, cells
were fixed by addition of cold 10% trichloroacetic acid (TCA, 4 °C)
and incubated at 4 °C for 1 h. After washing to remove TCA, drying
in air, 0.4% SRB dissolved in 1% acetic acid was added to each well
for staining at room temperature. Then, the plate was washed with
1% acetic acid to remove unbound SRB and dried again. Finally,
10 mM Tris base was added into each well to solubilize the cell-
bound dye. The plate was shaken followed by reading the optical
density (OD) at 515 nm using a microplate spectrophotometer.
of 100 lL horseradish peroxidase-conjugated goat anti-mouse
IgG (1:1000 diluted in 5 mg/mL BSA T-PBS) was added. The plate
was reincubated at 37 °C for 30 min, and washed as before. Finally,
3.3. Western blot analysis
100 lL of a solution containing 0.03% H2O2 and 2 mg/mL o-phenyl-
enediamine in 0.1 mM citrate buffer, pH 5.5, was added and sam-
ples were incubated at room temperature until color emerged.
The reaction was terminated by the addition of 50 lL of 2 M
Cells were cultured under regular growth conditions to the
exponential growth phase. The cells were then treated with appro-
priate concentrations of the corresponding compounds for 4 h at

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D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
Table 1
37 °C and lysed in 1 ꢀ SDS sample buffer. The lysates were subse-
quently resolved on 10% SDS-PAGE, and transferred to nitrocellu-
lose membranes. Membranes were probed with, phosphor-c-Met
and c-Met, phospho-ERK1/2 and ERK1/2, phospho-AKT and AKT
(All from Cell Signaling Technology, Beverly, MA) and GAPDH
(KangChen Bio-tech) antibody then subsequently with anti-rabbit
or anti-mouse IgG horseradish peroxidase. Immunoreactive pro-
teins were detected using an enhanced chemiluminescence detec-
tion reagent.
SAR of substituents on the amide bond
P 1
R1
R2
O
N
N
N
N
NH2
P 2
Compound
R1
H
R2
c-Met IC50
(lM)
EBC-1a IC50
NDb
(lM)
F
F
4. Results and discussion
10a
1.20 0.19
7.00 1.06
1-Methylpyrazole is usually used to modulate physical chemi-
cal properties in active c-Met inhibitors, presumably due to its
lower cLog P and higher PSA.42 In order to identify potent c-Met
inhibitors efficiently, we initially selected 1-methylpyrazole as a
substituent at the C-5 position (P2 part) while varying the amide
bond (P1 part) (Table 1). The initial biochemical assay found that
compounds 10a–10i exhibited moderate activity against c-Met
F
10b
H
NDb
F
F
F
10c
10d
H
H
0.60 0.03
4.11 0.04
15.00 1.05
F
F
with IC50 ranging from 7.00 to 0.41 lM. Clearly, derivatives bearing
2, 5-difluoro (10c) and 3,4-difluoro (10e) benzyl groups displayed
more potent c-Met inhibition than other difluoro substituted ana-
logs (10a, 10b, 10d). The activity did not significantly change with
the introduction of o,o0-disubstituents (10f–10i). Methylation of
the amide bond (16j-16l) resulted in the complete loss of activity,
suggesting that the N,N0- disubstituted amide was detrimental to
the binding affinity. Although the introduction of various benzyl
substituents on the amide bond gave no substantial change of
the binding affinity, the primary structure-activity relationship
(SAR) in Table 1 revealed that compounds (10c, 10e, 10h, 10i) dis-
played moderate enzymatic and EBC-1 cellular potency, warrant-
ing the further modification of the 2-aminopyridine-3-
carboxamide scaffold against c-Met kinase.
NDb
F
F
10e
H
0.48 0.06
14.58 2.35
10f
H
H
1.30 0.10
1.14 0.06
NDb
NDb
Cl
F
F
F
F
F
F
F
Cl
F
10g
We envisaged that the aryl fragments which had been intro-
duced on the amide bond (Table 1) lacked the effective p–p inter-
action with Y1230, which led us to install the 2,6-dichloro-3-
fluorobenzyl group (a key structural feature of crizotinib involved
F
10h
10i
H
0.41 0.10
0.56 0.06
6.90 0.82
12.71 1.82
NDb
Cl
Cl
Cl
in
p–p interaction with Y1230) with the expectation of an
improvement in potency. As anticipated, compound 24m (Table 2)
with the incorporation of this functionality exhibited an improved
IC50 of 0.28 lM, albeit suffering from a loss of cellular potency. The
encouraging result prompted us to optimize the substituents at the
H
Cl
F
16j
Me
Me
Me
10.9%@1
18.1%@1
13.1%@1
l
l
l
M
C-5 position. Appending the polar piperidine (24n) onto the pyra-
zole
retained
the
comparable
emzymatic
inhibition
F
(IC50 = 0.32
lM), and had a remarkable improvement in cellular
16k
M
NDb
potency. With the incorporation of a tetrahydropyranyl substitu-
ent, compound 24o exhibited a three-fold improvement of the
enzymatic potency compared with 24m, and also shown EBC-1
inhibition. Replacement of the pyrazole group with other hetero
(24p–24r) and aryl (24s) functionalities resulted in a sharp loss
of cellular potency. These observations indicated that modification
at the C-5 position had a significant influence on enzymatic and
cellular activity.
Cl
Cl
Cl
16l
M
NDb
Control
Crizotinib
0.0016 0.0002
0.053 0.011
a
EBC-1: human non-small-cell lung cancer cell line that expresses elevated
levels of constitutively active c-Met
b
Next, we wondered whether the amide bond might be too flex-
ible to enable the aryl groups linked with the amide bond effi-
ND: no data.
ciently involved in
p–p interaction with Y1230. To test this
hypothesis, compounds 29 and 30 in which the amide bond is
locked into the quinazolinone ring were prepared according to
Scheme 4. However, this strategy proved to be unsuccessful, prob-
ably due to the loss of an important H-bond between the amino
group and the hinge region (Table 3).
Given the promising enzymatic and cellular potency, compound
24o was selected for subsequent evaluation. In consideration of
configuration affecting the c-Met activity, the two enantiomers of
compound 24o were prepared. Biological evaluation found that
(S)-24o was more potent, with an enzymatic IC50 of 0.022 lM,
and an EBC-1 cell IC50 of 1.22 lM, respectively (Fig. 3). The effects
of the compound (S)-24o on c-Met phosphorylation and down-
stream signaling pathway were further evaluated in EBC-1 human
non-small-cell lung cancer cell line which expresses elevated levels
of constitutively active c-Met. We found that compound (S)-24o
inhibited the phosphorylation of c-Met in a dose-dependent
manner in EBC-1 cells. In addition, Erk1/2 and AKT, the key
downstream molecules of c-Met that play important roles in

D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
5173
Table 2
Table 4
SAR about the aryl/heroaryl derivatives at the C-5 position
Inhibition of tyrosine kinases by compound (S)-24o.
Kinase
Enzyme IC50 (lM)
F
RON
Mer
8.26 0.56
>10
P 1
Cl
Cl
TYRO3
AXL
KDR
EGFR
FGFR1
PDGFR
Flt-1
ABL
RET
EPH-A2
ErbB2
ErbB4
>10
>10
>10
>10
3.48 0.73
>10
8.16 0.38
4.99 3.95
0.21 0.09
6.18 2.86
>10
HN
Aryl
P 2
O
N
NH2
compound
Aryl
c-Met IC50
(lM)
EBC-1a IC50
> 50
(lM)
N
N
24m
0.28 0.04
0.32 0.08
N
>10
N
24n
1.46 0.07
HN
N
N
24o
24p
0.099 0.02
0.51 0.10
3.49 1.35
NDb
O
O
c-Met function, were also significantly inhibited upon treatment
with (S)-24o. These data support the fact that (S)-24o inhibits
c-Met activity as well as subsequent c-Met downstream signaling
at a cellular level.
To examine whether compound (S)-24o is a selective c-Met
inhibitor, (S)-24o was screened against another c-Met family
member Ron and other 13 tyrosine kinases (Table 4). Compared
N
N
H
N
24q
24r
0.21 0.03
1.14 0.03
17.80 1.34
NDb
O
N
N
to its high potency against c-Met (IC50 = 0.022 0.003 lM), (S)-
O
24o also exhibited inhibitory effects against RON, FGFR1 ABL,
EPH-A2, and Flt-1 although the potency was 2- to 9-fold lower than
that against c-Met. Moreover, compound (S)-24o showed little or
no kinase inhibition activity against other 7 tyrosine kinases
(IC50 > 10 lM) (Table 4). These data suggest that compound (S)-
24o is a promising selective multitarget kinase inhibitor.
O
N
24s
0.34 0.01
6.05 0.74
O
Control
Crizotinib
0.0016 0.0002
0.053 0.011
a
EBC-1: human non-small-cell lung cancer cell line that expresses elevated
levels of constitutively active c-Met
b
ND: no data.
5. Binding model analysis
Although the aminopyridine scaffold is frequently present in
c-Met inhibitors, the 2-aminopyridine-3-carboxamide scaffold
against c-Met has not been explored yet. To further elucidate the
binding mode of the novel 2-aminopyridine-3-carboxamide scaf-
fold with c-Met, a detailed docking analysis was performed. In
our study, the co-crystal structure of PF-02341066 with c-Met
was selected as the docking model (PDB ID code: 2WGJ). The con-
formation analysis was conducted using AutoDock4.2 due to its
powerful conformational search capabilities.48 For each compound,
50 conformations provided by the Lamarckian Genetic Algorithm
were obtained to efficiently probe the conformational binding
space (Fig. 4A). The binding model was exemplified by the interac-
tion of compound 24o with c-Met (Fig. 4C). Similar to crizotinib
(Fig. 4B), the amine and the nitrogen atom of the pyridine ring in
compound 24o H-bond well with the hinge protein residues
Pro1158 and Met1160. The pyrazole substituent extends into the
solvent-exposed region, indicating that the introduction of
Table 3
Effects of 3H,4H-pyrido[2,3-d]pyrimidin-4-ones on inhibitory activity against c-Met
O
N
Cl
F
Aryl
N
N
Cl
Compound
Aryl
c-Met IC50
28.9%@10
(lM)
N
N
29
l
M
M
BocN
HN
N
N
30
34.1%@10
l
F
F
Cl
Cl
Cl
Cl
N
N
N
HN
HN
N
O
O
O
O
N
)-24o
NH2
N
NH2
(
S
(R)-24o
c-Met 27.6%@1 μΜ
c-Met IC50 = 0.022 0.003 μΜ
EBC-1 IC50 = 1.22 0.20 μΜ
Figure 3. Effects of configuration of analog 24o on inhibitory activity and its downstream signaling pathways in EBC-1 cells.

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D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
Figure 4. (a) The structure of c-Met kinase was represented as cartoon, while compounds were shown as sticks in the pocket, with surrounding residues shown as gray
surface. (b) Binding poses of compounds (R)-pf-02341066 (gray stick) and (S)-pf-02341066 (purple stick). The surrounding key residues were shown in pink stick. (C) Binding
poses of compound (S)-24o (green stick) and (R)-24o (light red stick). (d) The estimated free energy of 50 conformations of compounds (S)-24o, (R)-24o, (S)-pf, (R)-pf. To be
clarity, the energy data was ranked. (e) The final total internal energy of the four compounds.
hydrophilic substituents here probably is beneficial for potency. In
addition, the pyridine and pyrazole rings are in a coplanar
conformation, stabilized by resonance, potentially increasing the
strength of the H-bond at the hinge region. Furthermore, the 2,6-
dichloro-3-fluorobenzyl group orientated by the amide function is
c-Met inhibitors. The SAR exploration led to the identification of
a potent compound (S)-24o with a c-Met IC50 of 0.022 M. This
l
compound exhibited dose-dependent inhibition of the phosphory-
lation of c-Met as well as c-Met downstream signaling in EBC-1
cells. Initial biochemical selectivity screening revealed that (S)-
24o is a multitarget kinase inhibitor. The interactive binding model
of (S)-24o was elucidated using AutoDock4.2. Further optimization
of the 2-aminopyridine-3-carboxamide scaffold in order to identify
compounds with improved enzymatic and cellular potency is cur-
rently under investigation.
involved in an excellent p-p interaction with residue Tyr1230.
In our study, our attention was drawn to the fact that the S enan-
tiomer of 24o is more potent than the R, while the situation for PF-
02341066 is vice versa. In addition to the optimized binding poses,
AutoDock4.2 has a free-energy scoring function based on a linear
regression analysis of a set of diverse protein-ligand complexes with
known inhibition constants. This scoring function uses the AMBER
force field to estimate the free energy of binding and final total
internal energy. Since the kinase protein was set rigid in the docking
simulation, the discrepancy on the total internal energy could be
theoretically viewed as the index of the internal energy of com-
pounds only, which may afford the clues for differentiated potency
here. Based on the estimated free energy of binding (Fig. 4D), it is
obvious that compound (R)-pf-02341066 is more potent than (S)-
pf-02341066, which is consistent with the observed value, indicat-
ing that the energetic scoring function is reasonable for this system.
In this model, compound (S)-24o does not have a significant advan-
tage compared with its enantiomer. Regarding the final total inter-
nal energy data (Fig. 4E), the 50 conformations of each compound
suggest that, in the binding pocket, (S)-24o has favorable internal
energy compared with (R)-24o, while (R)-pf-02341066 is more
advantageous than the S configuration. To be more specific, the
binding pose of (S)-24o is in a low energy conformation, which is
also the case for (R)-pf-02341066. In conclusion, owing to the acces-
sibility of the binding pose, different configurations in 24o and
pf-02341066 displayed different potency against the kinase.
7. Experimental
Unless otherwise noted, the reagents (chemicals) were pur-
chased from commercial sources, and used without further purifi-
cation. Analytical thin layer chromatography (TLC) was HSGF 254
(0.15-0.2 mm thickness). Compound spots were visualized by UV
light (254 nm). Column chromatography was performed on silica
gel FCP 200–300. NMR spectra were run on 300 or 400 MHz instru-
ment. Chemical shifts were reported in parts per million (ppm, d)
downfield from tetramethylsilane. Proton coupling patterns are
described as singlet (s), doublet (d), triplet (t), quartet (q), multi-
plet (m), and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were measured on spectrometer. Chiral HPLC
analysis was performed on a Chiral-Park IA column.
7.0.1. 2-Amino-5-bromonicotinic acid (8)
2-Aminonicotinic acid (10.00 g, 72.40 mmol) was suspended in
40 ml of glacial acetic acid. To this suspension was added dropwise
a solution of 4.82 ml (94.12 mmol) of bromine in 20 ml of glacial
acetic acid. The mixture was stirred vigorouly at room temperature
for 30 min. The formed precipitate was filtered off and washed
with glacial acetic acid. The filter cake was collected and crystal-
lized from the boiling methol to afford compound 8 as a white solid
6. Conclusions
In conclusion,
a series of 2-aminopyridine-3-carboxamide
derivatives were designed synthesized and evaluated as novel

D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
5175
(14.5 g, 92% yield). 1H NMR (300 MHz, DMSO-d6) d 8.30 (d, J =
7.0.8. 2-Amino-5-bromo-N-[(3-chloro-2,6-difluorophenyl)
methyl]pyridine-3-carboxamide (9g)
2.4 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H); MS (ESI, m/z): 216.8 [M+H]+.
Compound 9g was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
8.96 (t,
J = 4.8 Hz, 1H), 8.14 (d, J = 2.4 Hz, 1H), 8.09 (d, J = 2.4 Hz, 1H),
7.64–7.58 (m, 1H), 7.26 (s, 2H), 7.23–7.15 (m, 1H), 4.49 (d,
J = 4.8 Hz, 2H); MS (ESI, m/z): 375.9 [M+H]+.
7.0.2. 2-Amino-5-bromo-N-[(2,3-difluorophenyl)methyl]
pyridine-3-carboxamide (9a). General experimental procedure
for 9b-9i, 15j-15l and 22
d
2-Amino-5-bromonicotinic acid (250 mg, 1.15 mmol) was dis-
solved in 4 ml of DMF. The solution was cooled in an ice bath. Then
to the solution were successively added HATU (460 mg,
7.0.9. 2-Amino-5-bromo-N-[(2-chloro-3,6-difluorophenyl)
methyl]pyridine-3-carboxamide (9h)
1.21 mmol), DIPEA (210.68 ll, 1.21 mmol), (2,3-difluoro-
phenyl)methanamine (173 mg, 1.21 mmol). The resulting solution
was stirred at room temperature for 2 h and then the saturated so-
dium bicarbonate solution was added. The mixture was extracted
with ethyl acetate (EA). The combined organic layers were dried
over anhydrous Na2SO4 and concentrated under vacuum. The
crude product was used in the next step without purification. 1H
NMR (400 MHz, DMSO-d6) d 9.12 (t, J = 5.6 Hz, 1H), 8.16 (s, 2H),
7.38-7.09 (m, 5H), 4.49 (d, J = 5.6 Hz, 2H); MS (ESI, m/z): 341.8
[M+H]+.
Compound 9h was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
d 8.84 (t,
J = 4.4 Hz, 1H), 8.14 (d, J = 2.4 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H),
7.50–7.44 (m, 1H), 7.36–7.30 (m, 1H), 7.26 (s, 2H), 4.56 (d,
J = 3.6 Hz, 2H); MS (ESI, m/z): 375.8 [M+H]+.
7.0.10. 2-Amino-5-bromo-N-[(2,3-dichloro-6-fluorophenyl)
methyl]pyridine-3-carboxamide (9i)
Compound 9i was prepared in a similar manner as described for
compound 9a. 1H NMR (400 MHz, DMSO-d6) d 8.82 (t, J = 4.4 Hz,
1H), 8.14 (d, J = 2.4 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.69 (dd,
J = 5.6, 9.2 Hz, 1H), 7.35 (t, J = 9.2 Hz, 1H), 7.26 (s, 2H), 4.57 (d,
J = 3.2 Hz, 2H); MS (ESI, m/z): 391.9 [M+H]+.
7.0.3. 2-Amino-5-bromo-N-[(2,4-difluorophenyl)methyl]
pyridine-3-carboxamide (9b)
Compound 9b was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
d 9.06 (t,
J = 5.6 Hz, 1H), 8.16 (s, 2H), 7.46–7.40 (m, 1H), 7.35–7.15 (m, 3H),
7.09–7.01 (m, 1H), 4.42 (d, J = 5.6 Hz, 2H); MS (ESI, m/z): 341.9
[M+H]+.
7.0.11. 2-Amino-N-[(2,3-difluorophenyl)methyl]-5-(1-methyl-
1H-pyrazol-4-yl)pyridine-3-carboxamide (10a). General
experimental procedure for 10a–10i and 16j–16l
To
a
solution
of
2-amino-5-bromo-N-[(2,3-difluoro-
7.0.4. 2-Amino-5-bromo-N-[(2,5-difluorophenyl)methyl]
phenyl)methyl]pyridine-3-carboxamide 9a (250 mg, 0.73 mmol)
and 1-methyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
zole (160 mg, 1.05 mmol) was added sodium carbonate (232 mg,
2.19 mmol) solution in 1 ml of water, followed by Pd(PPh3)Cl2
(25.64 mg, 0.05 mmol). The resulting reaction medium was evacu-
ated and refilled with argon three times. Then the reaction medium
was heated at 80 °C for 16 h and was extracted with EA. After
washing with brine, the solution was dried over anhydrous Na2SO4
and concentrated under vacuum. The crude product was purified
by silica gel column chromatography (MeOH/CH2Cl2, 1/50) to af-
ford compound 10a as a white solid in 81% yield. 1H NMR
(400 MHz, CDCl3) d 8.24 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H),
7.61 (s, 1H), 7.52 (s, 1H), 7.20–7.06 (m, 3H), 6.83 (t, J = 5.6 Hz,
1H), 6.36 (s, 2H), 4.69 (d, J = 5.6 Hz, 2H), 3.90 (s, 3H); MS (ESI, m/
z): 344.0 [M+H]+; HRMS (ESI) calcd for C17H16F2N5O ([M+H]+):
344.1323; found : 344.1328.
pyridine-3-carboxamide (9c)
Compound 9c was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
d 9.08 (t,
J = 5.6 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H), 8.17 (d, J = 2.4 Hz, 1H),
7.30–7.09 (m, 5H), 4.44 (d, J = 5.6 Hz, 2H); MS (ESI, m/z): 342.0
[M+H]+.
7.0.5. 2-Amino-5-bromo-N-[(3,5-difluorophenyl)methyl]
pyridine-3-carboxamide (9d)
Compound 9d was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
d 9.13 (t,
J = 5.6 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H), 8.17 (d, J = 2.4 Hz, 1H),
7.27 (s, 2H), 7.14–7.01 (m, 3H), 4.44 (d, J = 5.6 Hz, 2H); MS (ESI,
m/z): 341.9 [M+H]+.
7.0.12. 2-Amino-N-[(2,4-difluorophenyl)methyl]-5-(1-methyl-
1H-pyrazol-4-yl)pyridine-3-carboxamide (10b)
7.0.6. 2-Amino-5-bromo-N-[(3,4-difluorophenyl)methyl]
pyridine-3-carboxamide (9e)
Compound 10b was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 47%; 1H NMR (400 MHz,
CDCl3) d 8.22 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.62 (s, 1H),
7.53 (s, 1H), 7.44–7.38 (m, 1H), 6.89–6.81 (m, 2H), 6.77 (br, 1H),
6.41 (s, 2H), 4.62 (d, J = 5.6 Hz, 2H), 3.90 (s, 3H). EI–MS m/z 343
[M]+; HRMS (EI) m/z calcd for [M]+ 343.1245, found 343.1238.
Compound 9e was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, DMSO-d6)
d 9.11 (t,
J = 5.6 Hz, 1H), 8.15 (s, 1H), 7.41–7.33 (m, 1H), 7.25 (s, 2H), 7.17–
7.06 (m, 1H), 4.40 (d, J = 5.6 Hz, 2H); MS (ESI, m/z): 341.7 [M+H]+.
7.0.7. 2-Amino-5-bromo-N-[(2-chloro-6-fluorophenyl)methyl]
pyridine-3-carboxamide (9f)
7.0.13. 2-Amino-N-[(2,5-difluorophenyl)methyl]-5-(1-methyl-
1H-pyrazol-4-yl)pyridine-3-carboxamide (10c)
Compound 9f was prepared in a similar manner as described for
compound 9a. 1H NMR (400 MHz, DMSO-d6) d 8.81 (t, J = 4.4 Hz,
1H), 8.14 (d, J = 2.4 Hz, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.46–7.22 (m,
5H), 4.54 (d, J = 3.6 Hz, 2H); MS (ESI, m/z): 357.9 [M+H]+.
Compound 10c was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 83%; 1H NMR (400 MHz,
DMSO-d6) d 9.04 (t, J = 5.6 Hz, 1H), 8.35 (d, J = 2.4 Hz, 1H), 8.18 (d,

5176
D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
J = 2.4 Hz, 1H), 8.01 (s, 1H), 7.79 (s, 1H), 7.30–7.24 (m, 1H), 7.22–
7.14 (m, 2H), 7.06 (s, 2H), 4.49 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H);
EI–MS m/z 343 [M]+; HRMS (EI) m/z calcd for [M]+ 343.1245, found
343.1250.
7.52 (s, 1H), 7.43 (dd, J = 5.6, 8.8 Hz, 1H), 7.05–7.01 (m, 1H), 6.68 (t,
J = 5.6 Hz, 1H), 6.33 (s, 2H), 4.82 (dd, J = 1.6, 5.6 Hz, 2H), 3.91 (s,
3H); EI–MS m/z 393 [M]+; HRMS (EI) m/z calcd for [M]+
393.0559, found 393.0559.
7.0.14. 2-Amino-N-[(3,5-difluorophenyl)methyl]-5-(1-methyl-
1H-pyrazol-4-yl)pyridine-3-carboxamide (10d)
7.1. tert-butyl N-[(3-chloro-2,6-difluorophenyl)methyl]
carbamate (12j). General experimental procedure for 12k–12l
Compound 10d was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 55%; 1H NMR (400 MHz,
CDCl3) d 8.28 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.61 (s, 1H),
7.52 (s, 1H), 6.90–6.86 (m, 2H), 6.81 (t, J = 5.6 Hz, 1H), 6.76–6.70
(m, 1H), 6.34 (s, 2H), 4.60 (d, J = 5.6 Hz, 2H), 3.90 (s, 3H); MS
(ESI, m/z): 344.0 [M+H]+; HRMS (ESI) calcd for C17H16F2N5O
([M+H]+): 344.1323; found : 344.1320.
Di-tert-butyl dicarbonate (385 mg, 1.76 mmol) was slowly
added to an ice-cooled stirred THF (4 ml) solution of (3-chloro-
2,6-difluorophenyl)methanamine (298 mg, 1.68 mmol). The result-
ing reaction mixture was stirred at 0 °C for 1 h. The reaction solu-
tion was concentrated under reduced pressure and the residue was
extracted with CH2Cl2. After washing with saturated NaCl, the or-
ganic layer was dried over anhydrous Na2SO4. After the removal
of the solvent by evaporation under vacuum, the crude product
was pure enough for use in the next step. 1H NMR (400 MHz,
DMSO-d6) d 7.60–7.54 (m, 1H), 7.36 (t, J = 5.6 Hz, 1H), 7.19–7.11
(m, 1H), 4.20 (d, J = 5.6 Hz, 2H), 1.36 (s, 9H).
7.0.15. 2-Amino-N-[(3,4-difluorophenyl)methyl]-5-(1-methyl-
1H-pyrazol-4-yl)pyridine-3-carboxamide (10e)
Compound 10e was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 77%; 1H NMR (400 MHz,
CDCl3) d 8.25 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 2.0 Hz, 1H), 7.61 (s, 1H),
7.52 (s, 1H), 7.20–7.06 (m, 3H), 6.85 (t, J = 5.6 Hz, 1H), 6.38 (s, 2H),
4.56 (d, J = 5.6 Hz, 2H), 3.89 (s, 3H); MS (ESI, m/z): 344.0 [M+H]+;
HRMS (ESI) calcd for C17H15F2N5O ([M+H]+): 344.1323; found :
344.1315.
7.2. tert-butyl N-[(2-chloro-3,6-difluorophenyl)methyl]
carbamate (12k)
Compound 12k was prepared in a similar manner as described
for compound 12j. 1H NMR (400 MHz, DMSO-d6) d 7.45–7.39 (m,
1H), 7.30–7.25 (m, 2H), 4.27 (d, J = 4.8 Hz, 2H), 1.36 (s, 9H).
7.0.16. 2-Amino-N-[(2-chloro-6-fluorophenyl)methyl]-5-(1-
methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide (10f)
7.3. tert-butyl N-[(2,3-dichloro-6-fluorophenyl)methyl]
carbamate (12l)
Compound 10f was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 53%; 1H NMR (400 MHz,
CDCl3) d 8.21 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.61 (d,
J = 1.6 Hz, 1H), 7.52 (s, 1H), 7.29–7.22 (m, 2H), 7.08–7.03 (m, 1H),
6.64 (t, J = 5.6 Hz, 1H), 6.39 (s, 2H), 4.80 (d, J = 1.6, 5.6 Hz, 2H),
3.92 (s, 3H); MS (ESI, m/z): 359.9 [M+H]+; HRMS (ESI) calcd for
Compound 12l was prepared in a similar manner as described
for compound 12j. 1H NMR (400 MHz, DMSO-d6) d 7.64 (dd,
J = 5.2, 8.8 Hz, 1H), 7.30–7.24 (m, 1H), 4.29 (d, J = 4.0 Hz, 2H),
1.36 (s, 9H).
7.4. tert-butyl N-[(3-chloro-2,6-difluorophenyl)methyl]-N-
methylcarbamate (13j). General experimental procedure for
13k–13l
C
17H16ClFN5O ([M+H]+): 360.1027; found : 360.1034.
7.0.17. 2-Amino-N-[(3-chloro-2,6-difluorophenyl)methyl]-5-(1-
methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide (10g)
69 mg (1.73 mmol) of sodium hydride (60 percent in oil) was
added portionwise to an ice-cooled solution of 400 mg
(1.44 mmol) of tert-butyl N-[(3-chloro-2,6-difluorophenyl)methyl]
carbamate in 4 ml of DMF, and the reaction medium was stirred for
Compound 10g was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 72%; 1H NMR (400 MHz,
CDCl3) d 8.24 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.61 (s, 1H),
7.52 (s, 1H), 7.37–7.31 (m, 1H), 6.93–6.88 (m, 1H), 6.68 (t,
J = 5.6 Hz, 1H), 6.33 (s, 2H), 4.73 (d, J = 5.6 Hz, 2H), 3.91 (s, 3H);
EI–MS m/z 377 [M]+; HRMS (EI) m/z calcd for [M]+ 377.0855, found
377.0850.
30 min. Then 183 ll (3.60 mmol) of methyl iodide was added drop-
wise and stirring is continued for 18 h. The ice-cold water was
added to the reaction medium. The mixture was extracted with
ethyl acetate. The organic phase was washed with water, brine,
dried over anhydrous Na2SO4 and concentrated under vacuum.
The crude product was purified by silica gel column chromatogra-
phy (EA/PE, 1/50) to afford compound 13j in 87% yield. 1H NMR
(400 MHz, CDCl3) d 7.34–7.28 (m, 1H), 6.88–6.83 (m, 1H), 4.53 (s,
2H), 2.85 (s, 3H), 1.46 (s, 9H).
7.0.18. 2-Amino-N-[(2-chloro-3,6-difluorophenyl)methyl]-5-(1-
methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide (10h)
Compound 10h was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 60%; 1H NMR (400 MHz,
CDCl3) d 8.27 (d, J = 2.4 Hz, 1H), 7.62 (s, 1H), 7.59 (d, J = 2.4 Hz, 1H),
7.51 (s, 1H), 7.16–7.10 (m, 1H), 7.06–7.01 (m, 1H), 6.52 (t,
J = 5.6 Hz, 1H), 6.25 (s, 2H), 4.81 (dd, J = 1.2, 5.6 Hz, 2H), 3.92 (s,
3H); EI–MS m/z 377 [M]+; HRMS (EI) m/z calcd for [M]+
377.0855, found 377.0848.
7.5. tert-butyl N-[(2-chloro-3,6-difluorophenyl)methyl]-N-
methylcarbamate (13k)
Compound 13k was prepared in a similar manner as described
for compound 13j. Yield: 94%; 1H NMR (400 MHz, CDCl3) d 7.11–
7.05 (m, 1H), 7.00–6.94 (m, 1H), 4.67 (s, 2H), 2.75 (s, 3H), 1.47 (s,
9H).
7.0.19. 2-Amino-N-[(2,3-dichloro-6-fluorophenyl)methyl]-5-(1-
methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide (10i)
7.6. tert-butyl N-[(2,3-dichloro-6-fluorophenyl)methyl]-N-
methylcarbamate (13l)
Compound 10i was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 64%; 1H NMR (400 MHz,
CDCl3) d 8.22 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H), 7.61 (s, 1H),
Compound 13l was prepared in a similar manner as described
for compound 13j. Yield: 90%; 1H NMR (400 MHz, CDCl3) d 7.40

D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
5177
(dd, J = 5.6, 8.8 Hz, 1H), 6.98 (t, J = 8.8 Hz, 1H), 4.69 (s, 2H), 2.74 (s,
3H), 1.47 (s, 9H).
8.16 (s, 1H), 7.64 (s, 1H), 7.61 (s, 1H), 7.55 (s, 1H), 7.19–7.14 (m,
1H), 7.08–7.03 (m, 1H), 5.90 (s, 2H), 4.94 (s, 2H), 3.95 (s, 3H),
2.91 (s, 3H); EI–MS m/z 391 [M]+; HRMS (EI) m/z calcd for [M]+
391.1011, found 391.1012.
7.6.1. [(3-Chloro-2,6-difluorophenyl)methyl](methyl)amine
(14j). General experimental procedure for 14k-14l, 24n and 30
To
a
solution of tert-butyl N-[(3-chloro-2,6-difluoro-
7.6.9. 2-Amino-N-[(2,3-dichloro-6-fluorophenyl)methyl]-N-
methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide
(16l)
Compound 16l was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 66%; 1H NMR (400 MHz,
CDCl3) d 8.23 (d, J = 2.0 Hz, 1H), 7.62 (d, J = 1.2 Hz, 1H), 7.51 (d,
J = 1.2 Hz ,1H), 7.48 (d, J = 2.4 Hz, 1H), 7.44 (dd, J = 5.6, 8.8 Hz,
1H), 7.04–7.01 (m, 1H), 5.19 (s, 2H), 4.94 (s, 2H), 3.92 (s, 3H),
2.87 (s, 3H); EI–MS m/z 407 [M]+; HRMS (EI) m/z calcd for [M]+
407.0716, found 407.0718.
phenyl)methyl]-N-methylcarbamate 13j (379 mg, 1.30 mmol) in
2 ml of CH2Cl2 was added 0.5 ml of TFA. The resulting solution
was stirred at room temperature for 2 h and concentrated under
vacuum. The residue was diluted with CH2Cl2 and washed with
saturated sodium carbonate, followed by brine. The organic layer
was dried over anhydrous Na2SO4. After the removal of the solvent
by evaporation under vacuum, the crude product was pure enough
for use in the next step. 1H NMR (400 MHz, CDCl3) d 7.32–7.25 (m,
1H), 6.87–6.82 (m, 1H), 3.90 (s, 2H), 3.75 (s, 1H), 2.42 (s, 3H).
7.6.2. [(2-chloro-3,6-difluorophenyl)methyl](methyl)amine
(14k)
Compound 14k was prepared in a similar manner as described
for compound 14j. 1H NMR (400 MHz, CDCl3) d 7.24–7.18 (m, 1H),
7.10–7.03 (m, 1H), 4.24 (d, J = 2.0 Hz, 2H), 2.62 (s, 3H).
7.6.10. 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-ol (18)
To a solution of 1-(2,6-dichloro-3-fluorophenyl)ethan-1-one 17
(20.00 g, 96.60 mmol) in 100 ml of MeOH was added portionwise
NaBH4 (7.31 g, 193.21 mmol). The resulting mixture was stirred
at room temperature for 2 h and then quenched with 20 ml of
water. After the removal of MeOH under vacuum, the residue
was extracted with EA and washed with brine. The organic layer
was dried over anhydrous Na2SO4. After the removal of the solvent
by evaporation under vacuum, the crude product was pure enough
for use in the next step. 1H NMR (400 MHz, CDCl3) d 7.26 (dd,
J = 4.8, 8.8 Hz, 1H), 7.02 (dd, J = 8.0, 8.8 Hz, 1H), 5.57 (q, J = 6.8 Hz,
1H), 2.86 (br, 1H), 1.64 (d, J = 6.8 Hz, 3H).
7.6.3. [(2,3-dichloro-6-fluorophenyl)methyl](methyl)amine
(14l)
Compound 14l was prepared in a similar manner as described
for compound 14j. 1H NMR (400 MHz, CDCl3) d 7.34 (dd, J = 5.2,
8.8 Hz, 1H), 6.95 (t, J = 8.8 Hz, 1H), 3.95 (d, J = 2.4 Hz, 2H), 2.38 (s,
3H).
7.6.4. 2-Amino-5-bromo-N-[(3-chloro-2,6-
7.6.11. 1-(2,6-Dichloro-3-fluorophenyl)ethyl methanesulfonate
(19)
difluorophenyl)methyl]-N-methylpyridine-3-carboxamide (15j)
Compound 15j was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, CDCl3) d 8.14 (s, 1H), 7.49
(s, 1H), 7.40–7.35 (m, 1H), 6.94–6.89 (m, 1H), 5.25 (s, 2H), 4.82
(s, 2H), 2.95 (s, 3H); MS (ESI, m/z): 389.9 [M+H]+.
To a solution of 1-(2,6-dichloro-3-fluorophenyl)ethan-1-ol 18
(19.00 g, 90.89 mmol) in 150 ml of CH2Cl2 was added Et3N
(13.27 ml, 95.43 mmol) and catalytic amount of DMAP. The result-
ing solution was cooled in an ice bath and added dropwise MsCl
(7.39 ml, 95.43 mmol). After complete addition of MsCl, the reac-
tion mixture was maintained in the ice bath for 1 h and then
30 ml of water was added to the reaction mixture. The organic
phase was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was pure enough
for use in the next step. 1H NMR (300 MHz, CDCl3) d 7.33 (dd,
J = 4.8, 9.0 Hz, 1H), 7.12 (dd, J = 8.1, 9.0 Hz, 1H), 6.45 (q, J = 6.9 Hz,
1H), 2.91 (s, 3H), 1.84 (d, J = 6.9 Hz, 3H).
7.6.5. 2-Amino-5-bromo-N-[(2-chloro-3,6-difluorophenyl)
methyl]-N-methylpyridine-3-carboxamide (15k)
Compound 15k was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, CDCl3) d 8.13 (d, J = 2.4 Hz,
1H), 7.48 (d, J = 2.0 Hz, 1H), 7.17–7.11 (m, 1H), 7.06–7.00 (m,
1H), 5.26 (s, 2H), 4.90 (s, 2H), 2.86 (s, 3H); MS (ESI, m/z): 389.8
[M+H]+.
7.6.6. 2-Amino-5-bromo-N-[(2,3-dichloro-6-fluorophenyl)
methyl]-N-methylpyridine-3-carboxamide (15l)
Compound 15l was prepared in a similar manner as described
for compound 9a. 1H NMR (400 MHz, CDCl3) d 8.14 (d, J = 2.4 Hz,
1H), 7.50–7.43 (m, 2H), 7.03 (t, J = 8.8 Hz, 1H), 5.27 (s, 2H), 4.91
(s, 2H), 2.85 (s, 3H); MS (ESI, m/z): 405.8 [M+H]+.
7.6.12. 2-(1-Azidoethyl)-1,3-dichloro-4-fluorobenzene (20)
To a solution of 1-(2,6-Dichloro-3-fluorophenyl)ethyl methane-
sulfonate 19 (500 mg, 1.74 mmol) in 4 ml of DMF was added NaN3
(226 mg, 3.48 mmol). The resulting mixture was stirred at 50 °C for
1 h and diluted with 20 ml of water. The mixture was extracted
with EA (2 ꢀ 10 ml). The organic phase was washed with brine,
dried over anhydrous Na2SO4 and concentrated under vacuum.
After the removal of the solvent by evaporation under vacuum,
the crude product was pure enough for use in the next step. 1H
NMR (400 MHz, CDCl3) d 7.31 (dd, J = 4.8, 8.8 Hz, 1H), 7.08 (dd,
J = 8.0, 8.8 Hz, 1H), 5.55 (q, J = 6.8 Hz, 1H), 1.65 (d, J = 6.8 Hz, 3H).
7.6.7. 2-Amino-N-[(3-chloro-2,6-difluorophenyl)methyl]-N-
methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide
(16j)
Compound 16j was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 42%; 1H NMR (400 MHz,
CDCl3) d 8.24 (d, J = 2.4 Hz, 1H), 7.64 (s, 1H), 7.52 (s, 1H), 7.49 (d,
J = 2.4 Hz, 1H), 7.37–7.33 (m, 1H), 6.93–6.88 (m, 1H), 5.12 (s, 2H),
4.84 (s, 2H), 3.93 (s, 3H), 2.97 (s, 3H); EI–MS m/z 391 [M]+; HRMS
(EI) m/z calcd for [M]+ 391.1011, found 391.1012.
7.6.13. 1-(2,6-Dichloro-3-fluorophenyl)ethan-1-amine (21)
2-(1-Azidoethyl)-1,3-dichloro-4-fluorobenzene 20 (300 mg,
1.28 mmol) was suspended in 4 ml of EtOH/H2O (v/v, 3/1). To this
suspension was added Zn powder (109 mg, 1.67 mmol) and NH4Cl
(171.40 mg, 3.20 mmol). The reaction mixture was heated at 80 °C
for 1 h. Then the reaction mixture was filtered off and concentrated
under vacuum. The residue was partitioned between ethyl acetate
and water. The organic phase was washed with brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude
product was pure enough for use in the next step. Yield: 71%; 1H
7.6.8. 2-Amino-N-[(2-chloro-3,6-difluorophenyl)methyl]-N-
methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide
(16k)
Compound 16k was prepared as a white solid in a similar man-
ner as described for compound 10a. Yield: 79%; (400 MHz, CDCl3) d

5178
D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
NMR (400 MHz, CDCl3) d 7.25 (dd, J = 4.8, 8.8 Hz, 1H), 6.99 (dd,
J = 8.0, 8.8 Hz, 1H), 4.90 (q, J = 6.8 Hz, 1H), 2.32 (s, 2H), 1.56 (d,
J = 6.8 Hz, 3H); MS (ESI, m/z): 208.0 [M+H]+.
(300 MHz, CDCl3) d 8.28 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 1.8 Hz, 1H),
7.69 (s, 1H), 7.60 (s, 1H), 7.32–7.25 (m, 1H), 7.17 (d, J = 8.4 Hz,
1H), 7.08–7.02 (m, 1H), 6.32 (s, 2H), 6.16–6.07 (m, 1H), 4.42–4.32
(m, 1H), 4.14–4.09 (m, 2H), 3.59–3.51 (m, 2H), 2.14–2.07 (m,
4H), 1.68 (d, J = 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) d 165.76,
158.53, 156.55, 156.09, 139.09, 136.11, 135.27, 129.96, 123.50,
118.44, 118.31, 116.22, 116.04, 111.68, 66.95, 58.67, 46.84, 33.48,
18.48; MS (ESI, m/z): 477.9 [M+H]+; HRMS (ESI) calcd for
7.7. 2-Amino-5-bromo-N-[1-(2,6-dichloro-3-fluorophenyl)
ethyl]pyridine-3-carboxamide (22)
Compound 22 was prepared in a similar manner as described
for compound 9a. 1H NMR (300 MHz, DMSO-d6)
d
9.12 (d,
C : 478.1213. The
22H23Cl2FN5O2 ([M+H]+): 478.1213; found
J = 5.4 Hz, 1H), 8.27 (d, J = 2.4 Hz, 1H), 8.15 (d, J = 2.4 Hz, 1H),
7.46 (dd, J = 5.1, 8.7 Hz, 1H), 7.36 (t, J = 8.7 Hz, 1H), 7.12 (s, 2H),
5.61–5.54 (m, 1H), 1.56 (d, J = 7.2 Hz, 3H); MS (ESI, m/z): 405.9
[M+H]+.
enantiomers of 24o were prepared by chiral resolution of the
racemic mixture on a chiral park IA column (1 cm ꢀ 25 cm,
EtOH/hexane = 1/3, flow rate: 5 mL/min, tR1 = 18.94 min, tR2
=
34.77 min,). The absolute stereochemistry of enantiomers was
determined by comparsion of HPLC retention times to (R)-24o,
which was prepared from the known (1S)-1-(2,6-dichloro-3-fluo-
rophenyl)ethan-1-ol as described in the Supplementary data
content.
7.8. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-(1-
methyl-1H-pyrazol-4-yl)pyridine-3-carboxamide (24m).
General experimental procedure for 24o–24s, 25 and 29
A vial was charged with 2-amino-5-bromo-N-[1-(2,6-dichloro-
7.12. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-[6-
3-fluorophenyl)ethyl]pyridine-3-carboxamide
22
(350 mg,
(morpholin-4-yl)pyridin-3-yl]pyridine-3-carboxamide (24p)
0.86 mmol), 1-methyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-pyrazole (179 mg, 0.86 mmol), KF.2H2O (243 mg, 2.58 mmol),
Pd(PPh3)4 (50 mg, 0.43 mmol) and 5 ml of DME/H2O/EtOH (v/v/v,
7/3/2). Then the vial was capped and heated at 110 °C for 50 min
under microwave irradiation. The reaction mixture was extracted
with EA. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated under vacuum. The crude
product was purified by silica gel column chromatography
(MeOH/CH2Cl2, 1/50) to afford compound 24m as a white solid.
Yield: 39%; 1H NMR (300 MHz, DMSO-d6) d 9.02 (d, J = 5.7 Hz,
1H), 8.34(d, J = 2.1 Hz, 1H), 8.22 (d, J = 2.1 Hz, 1H), 8.04 (s, 1H),
7.82 (s, 1H), 7.49 (dd, J = 5.1, 8.7 Hz, 1H), 7.40–7.34 (m, 1H), 6.89
(s, 2H), 5.69–5.60 (m, 1H), 3.39 (s, 3H), 1.62 (d, J = 7.2 Hz, 3H);
MS (ESI, m/z): 407.9 [M+H]+; HRMS (ESI) calcd for C18H17Cl2FN5O
[M+H]+: 408.0794, found: 408.0782.
Compound 24p was prepared as a white solid in a similar man-
ner as described for compound 24m. Yield: 40%; 1H NMR
(400 MHz, CDCl3)
d 8.34 (dd, J = 0.8, 2.4 Hz, 1H), 8.26 (d,
J = 2.4 Hz, 1H), 7.83 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 2.8, 8.8 Hz,
1H) 7.32–7.28 (m, 1H), 7.23 (d, J = 9.6 Hz, 1H), 7.05 (dd, J = 8.0,
8.8 Hz, 1H), 6.74 (dd, J = 0.4, 8.8 Hz, 1H), 6.60 (s, 2H), 6.19–6.09
(m, 1H), 3.86 (t, J = 8.8 Hz, 4H), 3.57 (t, J = 8.8 Hz, 4H), 1.69 (d,
J = 7.2 Hz, 3H); MS (ESI, m/z): 489.9 [M+H]+; HRMS (ESI) calcd for
C
23H23Cl2FN5O2 ([M+H]+): 490.1213; found : 490.1225.
7.13. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-(2-
oxo-2,3-dihydro-1H-indol-5-yl)pyridine-3-carboxamide (24q)
Compound 24q was prepared as a light red solid in a similar
manner as described for compound 24m. Yield: 20%; 1H NMR
(300 MHz, DMSO-d6) d 10.45 (s, 1H), 9.15 (d, J = 5.1 Hz, 1H), 8.35
(d, J = 2.1 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.55 (s, 1H), 7.50–7.45
(m, 2H), 7.36 (t, J = 8.7 Hz, 1H), 6.96 (s, 2H), 6.90 (d, J = 8.1 Hz,
1H), 5.68–5.59 (m, 1H), 3.56 (s, 2H), 1.60 (d, J = 7.2 Hz, 3H); MS
(ESI, m/z): 458.8 [M+H]+; HRMS (ESI) calcd for C22H18Cl2FN4O2
([M+H]+): 459.0791; found : 459.0778.
7.9. tert-butyl 4-[4-(6-amino-5-{[1-(2,6-dichloro-3-
fluorophenyl)ethyl]carbamoyl}pyridin-3-yl)-1H-pyrazol-1-
yl]piperidine-1-carboxylate (25)
Compound 25 was prepared in a similar manner as described
for compound 24m. Yield: 38%; 1H NMR (300 MHz, CDCl3) d 8.29
(d, J = 2.1 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.69 (s, 1H), 7.59 (s,
1H), 7.30 (dd, J = 5.1, 9.0 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.08–
7.03 (m, 1H), 6.24 (s, 2H), 6.16–6.11 (m, 1H), 4.35–4.25 (m, 3H),
2.96–2.87 (m, 2H), 2.18–2.15 (m, 2H), 2.03–1.89 (m, 2H), 1.69 (d,
J = 7.2 Hz, 3H), 1.49 (s, 9H); MS (ESI, m/z): 577.2 [M+H]+.
7.14. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-
(pyrimidin-5-yl)pyridine-3-carboxamide (24r)
Compound 24r was prepared as a white solid in a similar man-
ner as described for compound 24m. Yield: 37%; 1H NMR
(400 MHz, CDCl3) d 9.21 (s, 1H), 8.90 (s, 1H), 8.40 (d, J = 2.4 Hz,
1H), 7.84 (d, J = 2.4 Hz, 1H), 7.32–7.29 (m, 1H), 7.17 (d, J = 8.4 Hz,
1H), 7.06 (dd, J = 8.0, 8.8 Hz, 1H), 6.57 (s, 2H), 6.18–6.10 (m, 1H),
1.69 (d, J = 6.8 Hz, 3H); MS (ESI, m/z): 405.9 [M+H]+; HRMS (ESI)
calcd for C18H15Cl2FN5O ([M+H]+): 406.0638; found: 406.0645.
7.10. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-[1-
(piperidin-4-yl)-1H-pyrazol-4-yl]pyridine-3-carboxamide (24n).
Compound 24n was prepared in a similar manner as described
for compound 14j. Yield: 93%; 1H NMR (300 MHz, CDCl3) d 8.29 (d,
J = 2.1 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.69 (s, 1H), 7.61 (s, 1H), 7.30
(dd, J = 5.1, 9.0 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 8.1,
8.7 Hz, 1H), 6.24 (s, 2H), 6.19–6.08 (m, 1H), 4.31–4.23 (m, 1H),
3.31–3.27 (m, 2H), 2.86–2.77 (m, 2H), 2.23–2.19 (m, 2H), 2.03–
1.94 (m, 2H), 1.68 (d, J = 6.9 Hz, 3H); MS (ESI, m/z): 477.3
[M+H]+; HRMS (ESI) calcd for C22H24Cl2FN6O ([M+H]+): 477.1373;
found : 477.1361.
7.15. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-{4-
[2-(morpholin-4-yl)-2-oxoethoxy]phenyl}pyridine-3-
carboxamide (24s).
Compound 24s was prepared as a white solid in a similar man-
ner as described for compound 24m. Yield: 46%; 1H NMR
(400 MHz, CDCl3) d 8.34 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 2.4 Hz, 1H),
7.41 (d, J = 8.4 Hz, 2H), 7.31–7.28 (m, 1H), 7.19 (d, J = 8.8 Hz, 1H),
7.06–7.01 (m, 3H), 6.32 (s, 2H), 6.15–6.08 (m, 1H), 4.73 (s, 2H),
3.70–3.66 (m, 4H), 3.63–3.61 (m, 4H), 1.66 (d, J = 6.8 Hz, 3H); MS
(ESI, m/z): 546.9 [M+H]+; HRMS (ESI) calcd for C26H26Cl2FN4O4
([M+H]+): 547.1315; found : 547.1296.
7.11. 2-Amino-N-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-5-[1-
(oxan-4-yl)-1H-pyrazol-4-yl]pyridine-3-carboxamide (24o).
Compound 24o was prepared as a white solid in a similar man-
ner as described for compound 24m. Yield: 59%; 1H NMR

D. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 5169–5180
5179
7.16. 6-Bromo-3H,4H-pyrido[2,3-d]pyrimidin-4-one (26)
References and notes
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(1 g, 4.61 mmol) and 1.1 ml of formamide. The vial was capped
and heated at 150 °C for 30 min under microwave irradiation. To
the reaction mixture was added water. The precipitate was filtered
off and recrystallized from water to afford brown solid in 36% yield.
1H NMR (300 MHz, DMSO-d6) d 12.72 (s, 1H), 9.03 (d, J = 2.4 Hz, 1H),
8.61 (d, J = 2.4 Hz, 1H), 8.35 (s, 1H); MS (ESI, m/z): 225.8 [M+H]+.
3. Comoglio, P. M.; Giordano, S.; Trusolino, L. Nat. Rev. Drug Disc. 2008, 7, 504.
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7.17. 6-Bromo-3-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-3H,4H-
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pyrido[2,3-d]pyrimidin-4-one (27)
38.9 mg (0.97 mmol) of sodium hydride (60 percent in oil) was
added to an ice-cooled solution of 200 mg (0.88 mmol) of 6-bro-
mo-3H,4H-pyrido[2,3-d]pyrimidin-4-one 27 in 4 ml of DMF, and
the reaction medium is stirred for 10 min. Then 304.88 mg
(1.06 mmol) of 1-(2,6-dichloro-3-fluorophenyl)ethyl methanesul-
fonate 19 was added. The resulting mixture was heated at 80 °C
for 4 h. The ice-cold water was added to the reaction medium.
The mixture was extracted with ethyl acetate. The organic phase
was washed with water, brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude product was purified by sil-
ica gel column chromatography (EA/PE, 1/5-1/2) to afford com-
pound 27 in 50% yield. 1H NMR (300 MHz, CDCl3) d 8.99 (d,
J = 2.4 Hz, 1H), 8.69 (s, 1H), 8.61 (d, J = 2.4 Hz, 1H), 7.29–7.25 (m,
1H), 7.09–7.04 (m, 1H), 6.31 (q, J = 7.2 Hz, 1H), 2.01 (d, J = 7.2 Hz,
3H); MS (ESI, m/z): 415.7 [M+H]+.
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7.18. tert-butyl 4-(4-{3-[1-(2,6-dichloro-3-fluorophenyl)ethyl]-
4-oxo-3H,4H-pyrido[2,3-d]pyrimidin-6-yl}-1H-pyrazol-1-
yl)piperidine-1-carboxylate (29)
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Miknyoczki, S. J.; Murthy, S.; Rolon-Steele, D.; Underiner, T. L.; Wells-Knecht, K.
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23. Weinberg, L. R.; Albom, M. S.; Angeles, T. S.; Husten, J.; Lisko, J. G.; McHugh, R.
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T. L.; Zificsak, C. A.; Dorsey, B. D. Bioorg. Med. Chem. Lett. 2011, 21, 164.
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Pan, B.-S.; Marshall, C. G.; Lu, W.; Altman, M. D.; Dahlberg, W. K.; Davis, L.;
Falcone, D.; Gabarda, A. E.; Hang, G.; Hatch, H.; Holmes, R.; Kunii, K.; Lumb, K.
J.; Lutterbach, B.; Mathvink, R.; Nazef, N.; Patel, S. B.; Qu, X.; Reilly, J. F.; Rickert,
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Compound 29 was prepared in a similar manner as described
for compound 24m. Yield: 39.4%; 1H NMR (300 MHz, CDCl3) d
9.12 (d, J = 2.4 Hz, 1H), 8.65 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H), 7.85
(s, 1H), 7.78 (s, 1H), 7.29–7.25 (m, 1H), 7.09–7.03 (m, 1H), 6.34
(q, J = 7.5 Hz, 1H), 4.34–4.22 (m, 3H), 2.95–2.84 (m, 2H), 2.18–
2.13 (m, 2H), 2.03–1.86 (m, 5H), 1.47 (s, 9H); MS (ESI, m/z):
586.7 [M+H]+.
7.19. 3-[1-(2,6-Dichloro-3-fluorophenyl)ethyl]-6-[1-(piperidin-
yl)-1H-pyrazol-4-yl]-3H,4H-pyrido[2,3-d]pyrimidin-4-one (30)
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Compound 30 was prepared as a white solid in a similar as de-
scribed for compound 14j. Yield: 85%; 1H NMR (400 MHz, CD3OD) d
9.19–9.18 (m, 1H), 8.82 (s, 1H), 8.58–8.56 (m, 1H), 8.33 (s, 1H), 8.05
(s, 1H), 7.41-7.36 (m, 1H), 7.25–7.20 (m, 1H), 6.33–6.28 (m, 1H),
4.66–4.59 (m, 1H), 3.60–3.57 (m, 2H), 3.28–3.20 (m, 2H), 2.39–
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Acknowledgments
We gratefully acknowledge financial support from National Ba-
sic Research Program of China (Grants 2009CB940903 and
2012CB518000), the National Natural Science Foundation of China
(Grants 20721003 and 81025017), and National S&T Major Projects
(2012ZX09103-101-072).
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Supplementary data associated with this article can be found, in
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