Design, synthesis and biological evaluation of bis-aryl ureas and amides based on 2-amino-3-purinylpyridine scaffold as DFG-out B-Raf kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.10.039

By combining the scaffolds of UI-125 and Sorafenib, a series of bis-aryl ureas and amides based on 2-amino-3-purinylpyridine moiety were designed and synthesized as novel DFG-out B-Raf^V600E inhibitors. Among them, 20c-e, 20g and 21h displayed potent antiproliferative activities against melanoma A375 (B-Raf^V600E) cell lines with IC₅₀ values of 3.190, 2.276, 1.856, 1.632 μM and 1.839 μM, respectively, comparable with the positive control Vemurafenib (IC₅₀ Combining double low line 3.32 μM). Selected compounds were tested for the ERK inhibition in human melanoma A375 (B-Raf^V600E) and SK-MEL-2 (B-Raf^WT) cell lines by Western blot. The results revealed that our compounds inhibited the proliferation of melanoma A375 cells (B-Raf^V600E) through ERK pathway, without paradoxical activation of ERK in melanoma SK-MEL-2 cells (B-Raf^WT). Eventually, 20g and 21h were selected to confirm their inhibitory effects on tumor growth in A375 xenograft models in mice. Compound 20g exhibited equivalent antitumor efficacy in vivo (T/C Combining double low line 44.37%), compared to Sorafenib (T/C Combining double low line 37.35%), by 23-day repetitive administration of a single dose of 50 mg/kg without significant body weight loss.

Full text of DOI:10.1016/j.ejmech.2014.10.039

European Journal of Medicinal Chemistry 89 (2015) 581e596
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of bis-aryl ureas and
amides based on 2-amino-3-purinylpyridine scaffold as DFG-out B-Raf
kinase inhibitors
Weimin Yang a, Yadong Chen a, Xiang Zhou a, Yazhou Gu a, Wenqi Qian a, Fan Zhang a,
,
b
,
, **
Wei Han a, Tao Lu a *, Weifang Tang a
a School of Basic Sciences, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu 211198, China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
By combining the scaffolds of UI-125 and Sorafenib, a series of bis-aryl ureas and amides based on 2-
amino-3-purinylpyridine moiety were designed and synthesized as novel DFG-out B-RafV600E in-
hibitors. Among them, 20cee, 20g and 21h displayed potent antiproliferative activities against mela-
Received 17 March 2014
Received in revised form
14 October 2014
Accepted 14 October 2014
Available online 16 October 2014
noma A375 (B-RafV600E) cell lines with IC50 values of 3.190, 2.276, 1.856, 1.632
m
M and 1.839
mM,
respectively, comparable with the positive control Vemurafenib (IC50 ¼ 3.32
mM). Selected compounds
were tested for the ERK inhibition in human melanoma A375 (B-RafV600E) and SK-MEL-2 (B-RafWT) cell
lines by Western blot. The results revealed that our compounds inhibited the proliferation of melanoma
A375 cells (B-RafV600E) through ERK pathway, without paradoxical activation of ERK in melanoma SK-
MEL-2 cells (B-RafWT). Eventually, 20g and 21h were selected to confirm their inhibitory effects on tu-
mor growth in A375 xenograft models in mice. Compound 20g exhibited equivalent antitumor efficacy
in vivo (T/C ¼ 44.37%), compared to Sorafenib (T/C ¼ 37.35%), by 23-day repetitive administration of a
single dose of 50 mg/kg without significant body weight loss.
Keywords:
B-Raf inhibitors
DFG-Out
Melanoma
Antitumor agents
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Accumulating compounds have been developed as antitumor
agents targeting B-Raf and categorized into two types: DFG-in and
As significant players of the conserved MAPK cascade
(RaseRafeMEKeERK signaling pathway), Raf kinases including A-
Raf, B-Raf and C-Raf (Raf-1), are critical in mediating cell prolifer-
ation and survival [1]. B-Raf displays more potential biochemical
activity than A-Raf and Raf-1 [2]. Furthermore, over 40 different
mutations in B-Raf kinase domain have been identified to date, in
contrast to rare mutations found in A-Raf and Raf-1 [2]. Over 90% of
aberration in B-Raf is a substitution of a glutamic acid for valine at
residue 600 (V600E) [3], which is particularly associated with
melanoma and occurs with a frequency of 60% [4]. Based on
epidemiology and preclinical target conformation, B-Raf has been
recognized as an attractive target for cancer treatment [5].
DFG-out, according to their binding modes derived from crystallo-
graphic analysis and molecular modeling [2,6,7]. The DFG-in in-
hibitors, such as Vemurafenib (1) and Dabrafenib (2), bind to the ATP
binding site of the kinase with a DFG-in “active” conformation and
exhibit potential and highly selective inhibition against B-RafV600E
(Fig.1). The DFG-out inhibitors such as Sorafenib (3) and RAF265 (4),
engage the protein stable in DFG-out conformation (Fig.1). Recently,
the rapid development of secondary malignancies is reported as a
well-known side effect among those selective B-RafV600E inhibitors
[8e12] due to the paradoxical stimulation of MAPK signaling in cells
bearing wild-type B-Raf (B-RafWT) and Raf-1 [13,14]. It is demon-
strated that the feedback activation in B-RafWT bearing cancers can
be significantly suppressed by DFG-out Raf inhibitors, but not
by DFG-in type [15]. Hence, an optimal B-Raf inhibitor should
be DFG-out type that preserves potency against oncogenic B-Raf
without driving hyperactivation of B-RafWT or Raf-1.
* Corresponding author. State Key Laboratory of Natural Medicines, China Phar-
maceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China.
** Corresponding author.
E-mail addresses: lutao@cpu.edu.cn, lut163@163.com (T. Lu), twf@cpu.edu.cn
(W. Tang).
UI-152 (5, Fig. 2) has been disclosed as a potent and selective
ATP-competitive B-Raf inhibitor with an IC50 value of 1 nM [16].
Like other oncogenic B-Raf inhibitors, UI-152 has also been
http://dx.doi.org/10.1016/j.ejmech.2014.10.039
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
Fig. 1. Structures of Vemurafenib (1), Dabrafenib (2), Sorafenib (3) and RAF265 (4).
Fig. 2. Chemical structures of UI-152, compound 6a and target compounds.
reported to cause the paradoxical activation of the MAPK pathway
through activating phosphorylation of downstream ERK kinase in
B-RafWT bearing cancers [16]. Our current work demonstrated that
a novel DFG-out inhibitor 6a could be evolved from UI-152 by
replacing the furan-3-sulfonamide moiety with bis-aryl urea frag-
ment, while remaining the 2-amino-3-purinylpyridine core intact
(Fig. 2). Subsequent optimization of 6a focused on the ring
Aelinkerering B domain and provided a series of 2-amino-3-
purinylpyridine scaffold based derivatives for further biological
evaluation and structureeactivity relationship (SAR) exploration
(Fig. 2). Downstream inhibitions of ERK phosphorylation (p-ERK)
were performed in both B-RafV600E mutant and B-RafWT harboring
melanoma cells lines. In vivo studies in a human melanoma A375
xenograft model were also investigated in our work.
2. Results and discussion
2.1. Inhibitors design and molecular modeling
6a was employed as a starting point for further optimization to
develop more attractive and potent compounds. We first changed
the linker between phenyl ring A and B from urea (NHCONH) to

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
583
amide (NHCO) and “reverse” amide (CONH) based upon the pre-
viously known B-Raf inhibitor designs [17e19]. Then the replace-
ment of R1 at C-4 position on phenyl ring B with different lipophilic
groups was attempted to investigate their effects on kinase activity
(Fig. 3). Finally, the terminal phenyl ring A was substituted with a
variety of other functionalities for further optimization. A two-
dimensional pharmacophore map depicted the interaction be-
tween our compounds and B-RafV600E in a simplified form shown in
Fig. 3. From this pharmacophore model, we can speculate that: (1)
the purine ring is significant to form hydrogen bonds with Cys532
of the hinge region; (2) either urea or amide as linkers form the
the room temperature for about 3e6 h to generate the aniline
products 9aeb, 13aeo and 16aeh.
The synthetic route of target compounds 6aeb, 20aeo and
21aeh is outlined in Scheme 2. The commercially available 6-
chloro-9H-purine 17 was converted to 6-chloro-9-(tetrahydro-2H-
pyran-2-yl)-9H-purine 18 according to a reported method with
minor modification [21]. Subsequently, the reaction of 18 with 2-
fluoro-3-pyridylboronic acid through a SuzukieMiyaura coupling
provided 19 [22]. Then, 19 were converted to 6aeb, 20aeo and
21aeh through SNAr reaction with the previously prepared
different substituted amines 9aeb, 13aeo and 16aeh (Scheme 2)
using lithium bis(trimethylsilyl)amide (LiHMDS) as a base in
tetrahydrofuran (THF) in reflux condition, followed by removal of
the THP protecting group under acidic conditions to afford target
compounds 6aeb, 20aeo and 21aeh.
required hydrogen bonds with the conserved Glu501 of the
aC-
helix and the backbone amide of Asp594 from the DFG motif; (3) R1
group on phenyl ring B occupies a small hydrophobic pocket typi-
cally closed off the gatekeeper region; (4) the R2 substituted phenyl
ring A is exposed to the hydrophobic back pocket which is made
accessible by a rearrangement of the activation loop and subse-
quent movement of a phenylalanine side chain of DFG loop [20].
Based on these information, a library of 25 novel inhibitors were
designed and then synthesized.
2.3. Biological evaluation
2.3.1. B-RafV600E inhibitory assay
The newly synthesized compounds were evaluated as B-
RafV600E inhibitors using Sorafenib and Vemurafenib as positive
reference compounds. As shown in Table 1, all compounds showed
superior potency to Sorafenib (IC50 ¼ 26.4 nM) and Vemurafenib
(IC50 ¼ 23.0 nM) with IC50 values at nanomolar ranges. Particularly,
compounds 20e (IC50 ¼ 0.717 nM), 21d (IC50 ¼ 0.669 nM) and 21h
(IC50 ¼ 0.790 nM) showed remarkable potency with IC50 values less
than 1 nM.
2.2. Chemistry
A total of 25 important intermediates of anilines 9aeb, 13aeo
and 16aeh were synthesized according to Scheme 1 via a two-step
process. Urea analogs 8a and 8b were generated from 4-methyl-3-
nitroaniline (7a) and 3-nitroaniline (7b) with 1-chloro-4-
isocyanato-2-(trifluoromethyl)benzene, while amide analogs
12aeo and 15aeh were obtained by condensation of benzoic acid
10aed and 14aeg with their corresponding anilines 11aej and
7aeb listed in Scheme 1. The amide-forming reactions were carried
out in the presence of EDCI and HOBt in anhydrous DMF at the
room temperature for 4e24 h. Then, reduction of the nitro group
for 8aeb, 12aeo and 15aeh was carried out by using 5% Pd/C in
ethanol/tetrahydrofuran (1:1) under hydrogenation conditions at
2.3.2. Antiproliferative assay against A375 cells in vitro
As described above, B-RafV600E was an important target for
developing small-molecular inhibitors for cancer therapies, espe-
cially melanoma. The antiproliferative activities of all synthesized
compounds against A375 cells (B-RafV600E) were evaluated and the
results were summarized in Table 1. Compounds 20cee, 20g and
21h (IC50 ¼ 3.190, 2.276,1.856,1.632
mM and 1.839 mM, respectively)
displayed potent antiproliferative activities against A375 cell line,
comparable
with
M). Almost half of the compounds proved to be su-
M) but less potent than Vemur-
the
positive
control
Vemurafenib
(IC50 ¼ 3.315
m
perior to Sorafenib (IC50 ¼ 13.64
m
afenib. The rest of tested compounds showed weak B-Raf inhibition
with IC50 values range of 16.01e79.50 M.
m
2.3.3. Structureeactivity relationship exploration
All synthesized compounds showed distinct enzymatic and
cellular inhibition due to the difference of R1, R2 and linker types (L)
between two phenyl rings.
2.3.3.1. SAR of linker types (L) between the phenyl ring A and B.
Replacement of the urea linker of 6a with amide and “reverse”
amide afforded compounds 20a (L ¼ NHCO) and 21a (L ¼ CONH),
respectively. Compared to 6a (B-RafV600E IC50 ¼ 12.0 nM, A375
IC50
IC50 ¼ 61.35
significantly reduced cellular activity, while 21a (B-RafV600E
¼
27.27
m
M), 20a (B-RafV600E IC50
¼
13.5 nM, A375
m
M) got a slight loss in inhibitory potency and a
IC50 ¼ 3.03 nM, A375 IC50 ¼ 16.01
mM) exhibited a 4-fold
enhancement in B-RafV600E inhibition with attendant improve-
ment in cellular potency. However, the methyl substituted amide
20b (B-RafV600E IC50 ¼ 3.15 nM, A375 IC50 ¼ 8.785
mM) and
“reverse” amide 21b (B-RafV600E IC50
¼
2.71 nM, A375
IC50 ¼ 8.42
mM) were more potent than the corresponding urea
analog 6b (B-RafV600E IC50 ¼ 4.21 nM, A375 IC50 ¼ 29.73
mM) in both
B-RafV600E and A375 inhibition. These results suggested that both
amide (L ¼ NHCO) and “reverse” amide (L ¼ CONH) were tolerant to
the potency. They could be both attributed to hydrogen bond for-
mation at the specific receptor sites like the urea linker as we
Fig. 3. Structural modification of 2-amino-3-purinylpyridine derivatives and the two-
dimensional pharmacophore map of binding mode to the DFG-out conformation of B-
Raf.

584
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
Scheme 1. The synthesis of aniline intermediates 9aeb, 13aeo and 16aeh. Reagents and conditions: (a) 1-chloro-4-isocyanato-2-(trifluoromethyl)benzene, CH2Cl2, rt, 2 h; (b) H2, 5%
Pd/C, EtOH/THF (1:1), rt, 3e6 h; (c) EDCI, HOBt, Et3N, DMF, rt, 4e24 h.
Scheme 2. Synthesis of target compounds 6aeb, 20aeo and 21aeh. Reagents and conditions: (a) 3,4-dihydro-2H-pyran, TsOH, anhydrous EtOAc, reflux, 3 h; (b) (2-fluoropyridin-3-
yl)boronic acid, PdCl2(dppf), Na2CO3, 1,4-dioxane/H2O (4:1), nitrogen atmosphere, reflux, 14 h; (c) 9aeb, 13aeo and 16aeh, LiHMDS, anhydrous THF, nitrogen atmosphere, rt, 2e6 h;
(d) 1 N HCl, MeOH, rt, 2 h.
estimated (Fig. 3). Further evaluation of 20bej and 21beh revealed
that the amide derivatives (L ¼ NHCO) shared fairly comparable
potency with “reverse” amide analogs (L ¼ CONH) against B-
RafV600E and A375 cells.
2.3.3.2. SAR of substitutions R1 on the ring B. Subsequently, the
influence of R1 substituent at C-4 position on ring B was probed.
Compounds 6b, 20b and 21b with methyl substitution displayed
enhanced B-Raf kinase inhibition, as well as A375 antiproliferative

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
585
Table 1
The list of bis-aryl urea and amide analogs based on 2-amino-3-purinylpyridine scaffold and biological evaluation against B-Raf and melanoma A375 cells.
Compounds
L
R1
R2
IC50 values
B-Rafa (nM)
A375b,c
(mM)
6a
6b
NHCONH
NHCONH
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
NHCO
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
H
CH3
H
4-Cl-3-CF3
4-Cl-3-CF3
4-Cl-3-CF3
4-Cl-3-CF3
3-CF3
3-CH(CH3)2
3-CN
3-F
4-Cl-3-CH3
3-Cl
3-Br-4-Cl
4-CF3
4-Cl-3-CF3
3-CF3
3-CH(CH3)2
3,4-di-Cl
4-Cl-3-CF3
4-Cl-3-CF3
4-Cl-3-CF3
3-CF3
3-CN
4-Cl-3-CH3
3-Cl
12.0
4.21
13.5
3.15
2.17
1.69
0.717
1.03
1.43
1.30
3.62
1.50
10.8
2.78
8.07
1.60
4.82
3.03
2.71
2.51
0.669
2.56
1.77
1.20
0.790
27.27 17.055
29.73 6.89
61.35 39.71
8.785 2.300
3.190 0.751
2.276 0.773
1.856 0.47
79.50 55.27
1.632 0.482
6.285 1.6
10.01 3.054
10.03 2.11
13.28 4.212
11.43 3.242
7.512 1.444
51.95 30.325
19.97 3.98
16.01 3.96
8.42 1.898
7.17 1.493
10.62 3.279
6.914 1.761
10.75 2.866
3.718 1.19
1.839 0.572
20a
20b
20c
20d
20e
20f
20g
20h
20i
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
20j
20k
20l
Cl
Cl
Cl
F
20m
20n
20o
21a
21b
21c
21d
21e
21f
21g
21h
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3-OCH3
3-N(CH3)2
22
23
24
7.44
68.83 33.68
471
>100
6430
>100
Sorafenib
Vemurafenib
26.4
23.0
13.64 3.045
3.315 0.58
a
B-RafV600E inhibitory assay.
Antiproliferative assay in A375 cells.
Values are expressed as means SD (standard deviation) of five experiments.
b
c
potency than the corresponding unsubstituted compounds 6a, 20b
and 21b. The comparison indicated that the methyl group in R1
position was beneficial for the potency. This result was consistent
with the hypothesis that the R1 group would occupy a small hy-
drophobic pocket typically closed off the gatekeeper region in B-
Raf. Furthermore, halogen atoms, including fluorine and chloride,
were also employed to afford compounds 20keo. However, these
halogen-substituted compounds displayed obviously less inhibi-
tory performances than the methyl substituted derivatives 20bed.
The strength order against B-RafV600E is CH3 > F > Cl > H, while the
potency order against A375 is CH3 > Cl > F > H. These results proved
that C-4 substitution of methyl group on phenyl ring B was a fruitful

586
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
Fig. 4. Compounds 20cee, 20g and 21h potently inhibits ERK phosphorylation. A375 (A) or SK-MEL-2 (B) human malignant melanoma cells were treated with the indicated
concentrations of our compounds or reference compound PLX4032 (starting at 1 M, 7 doses, 3 fold dilution) for 90 min. The ERK phosphorylation (p-ERK) and expression levels (t-
ERK) were analyzed by Western blotting with anti-phospho-ERK antibody or anti-ERK antibody. (C) Summary of the IC50 values by Western blot assay.
m
approach to enhance B-RafV600E inhibition relative to melanoma
A375 cells antiproliferative activity.
the binding affinity between the terminal phenyl ring A and the
hydrophobic pocket.
2.3.3.3. SAR of substitutions R2 on the ring A. Different lipophilic
substitutions were attempted at the meta and (or) para positions of
terminal phenyl ring A (R2) to determine their effects on potency,
affording derivatives 20cej and 21ceh. Apart from 4-chloro-3-
bromo substituent found in compounds 20i, both electron with-
drawing groups (like halogen, cyano) and electron donating groups
(like methoxy, dimethylamino) were more accommodated than the
initial 4-chloro-3-trifluoromethyl group. Compounds 20e
(IC50 ¼ 0.717 nM), 21d (IC50 ¼ 0.669 nM) and 21h (IC50 ¼ 0.790 nM)
disclosed the best kinase inhibition with IC50 values less than 1 nM,
while compounds 20cee, 20g and 21h exhibited the highest po-
tency against human melanoma A375 cell line with IC50 values of
2.3.4. ERK kinase inhibition in A375 cells
In order to explore the biological mechanism of these DFG-out
compounds, 20cee, 20g and 21h with high potency against hu-
man melanoma A375 cell line were selected as representative ex-
amples to be tested for their inhibitory effects on ERK kinases using
reported Western blot methods [15,23,24]. A375 cell lysate was
treated with seven different concentrations (1 mM as starting con-
centration with 3 fold dilution) of the test compounds, and their
inhibitory activities were compared with that of Vemurafenib
(PLX4032). The results displayed as Western blot band signals in
Fig. 4A, indicating that our compounds and Vemurafenib markedly
suppressed phosphorylation of ERK1/2 in a dose dependent
manner. Furthermore, the IC50 values of ERK phosphorylation in
A375 cells of these five compounds was listed in Fig. 4C, suggesting
that all compounds exhibited much more strong inhibitory effects
quantitatively on the ERK kinase than Vemurafenib. Therefore, the
result validated the hypothesis that these compounds may inhibit
the proliferation of melanoma cells harboring B-RafV600E mutation
through ERK pathway. The observed activities were not due to off-
target effects on proliferation but B-Raf inhibition.
3.190, 2.276, 1.856, 1.632 mM and 1.839 mM, respectively. These re-
sults suggested that 3-trifluromethyl, 3-isopropyl, 3-cyano, 4-
chloro-3-methyl and 3-dimethylamino groups were beneficial for
potency. However, 3-fluro group found in compound 20f (B-
RafV600E IC50 ¼ 1.03 nM, A375 IC50 ¼ 79.50
mM) was unfavorable due
to the 9-fold reduction of antiproliferative potency, compared with
20b (A375 IC50 ¼ 8.785
mM).
2.3.3.4. SAR of purine ring and terminal phenyl ring A. A methyl
group was added onto the 9-NH of purine ring of 20d (B-RafV600E
2.3.5. ERK kinase inhibition in SK-MEL-2 cells
IC50 ¼ 1.69 nM, A375 IC50 ¼ 2.276
m
M), affording compound 23 (B-
Reflecting the potent B-RafV600E inhibitory activities in vitro,
compounds 20g and 21h inhibited phosphorylation of ERK (p-ERK)
in melanoma A375 cells with IC50 values of 7.13 nM and 6.31 nM,
respectively (Fig. 4C). Subsequently, the inhibition of ERK phos-
phorylation (p-ERK) were also evaluated against melanoma SK-
MEL-2 cells expressing B-RafWT, which have been reported to be
insensitive to selective B-RafV600E inhibitors such as Vemurafenib.
The results displayed as Western blot band signals in Fig. 4B.
Vemurafenib was observed to activate the signal transduction of
the MAPK cascade through phosphorylation of ERK in a dose-
dependent manner in SK-MEL-2 cells. However, compounds 20g
and 21h were assessed to inhibit the phosphorylation of ERK (p-
ERK) potently with IC50 values of 15.9 nM and 5.12 nM, respectively
(Fig. 4C). These results showed that the bis-aryl ureas and amides
RafV600E IC50 ¼ 7.44 nM, A375 IC50 ¼ 68.83
mM), which resulted in
a 34-fold loss of antiproliferative activity (Table 1). The synthetic
route and procedure of compound 22 were listed in
Supplementary data. This result confirmed that the 9-NH on pu-
rine ring was significant to remain the hydrogen bond interactions
with hinge residues of B-RafV600E. Meanwhile, terminal aromatic
ring A were replaced into non-aromatic fragments such as cyclo-
propyl ring and methyl group to generate compound 23 (B-
RafV600E IC50 ¼ 471 nM, A375 IC50 > 100
m
M) and 24 (B-RafV600E
mM, A375 IC50 > 100 mM), respectively. They were also
IC50 ¼ 6.43
synthesized (listed in Supplementary data) and evaluated, but
almost inactive in both kinase and cellular inhibition (Table 1). The
bis-aryl ring structure is estimated significant for the potency and

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
587
Fig. 5. Antitumor activities of 20g and 21h (i.g.) in human melanoma A375 (B-RafV600E) bearing Balb/c-nu nude mice (A) Mean with SEM values (n ¼ 6; *P ꢁ 0.05 vs Vehicle) of
tumor volumes according to days after the treatment of Sorafenib, 20g, 21h and vehicle group. (B) Mean with SEM values (n ¼ 6; *P ꢁ 0.05 vs Vehicle) of body weight according to
days after the treatment of Sorafenib, 20g, 21h and vehicle group. (C) The photograph of tumors from dissected mice at the end of experiment. (D) Summary of doses, T/C values and
body weight changes in A375 (B-RafV600E) xenograft models.
based on 2-amino-3-purinylpyridine moiety would apparently
suppress the proliferation of B-RafV600E positive melanoma cell
lines without activation of the MAPK signaling in B-RafWT bearing
cells. The most representative compounds 20g and 21h could be
identified as promising antitumor agents to treat melanoma pa-
potent antiproliferative activities against A375 cell line with IC50
values of 3.190, 2.276, 1.856, 1.632 M and 1.839 M, respectively,
m
m
were selected for further evaluation of the p-ERK inhibition in
human melanoma A375 (B-RafV600E) and SK-MEL-2 (B-RafWT) cells.
A key finding from this class of compounds was that, unlike
PLX4032, they dramatically inhibited the proliferation of mela-
noma A375 cells (B-RafV600E) through ERK pathway, without par-
adoxical activation of the MAPK in SK-MEL-2 (B-RafWT) cells. The
most potent compounds 20g and 21h were progressed to in vivo
profiling. 20g exhibited similarly equivalent tumor growth inhibi-
tion (T/C ¼ 44.37%) by contrast to Sorafenib (T/C ¼ 37.35%) with no
significant body weight loss. The further study will focus on the
physicochemical property improvement and druggabilityoptim-
ization of these compounds, for the poor water-solubility was
observed during the pharmacological experiments, which is sup-
posed to dramatically affect the inhibition of tumor growth in vivo.
tients with either B-RafV600E or B-RafWT
.
2.3.6. In vivo studies of compounds 20g and 21h in Balb/c-nu nude
mice
To preliminarily examine the efficacy of 20g and 21h in vivo, the
two compounds were prepared in solid dispersion (SD) formulation
and intragastrically administered once daily for 23 days over a
single dose of 50 mg/kg in a human melanoma A375 (B-RafV600E
)
xenograft model in Balb/c-nu nude mice (Fig. 5). As a result, com-
pound 21h displayed obvious antitumor efficacy (T/C ¼ 71.53%)
compared with the vehicle control and showed no signs of body
weight loss (body weight change: þ1.92 g) (Fig. 5D). In particular,
compound 20g exhibited equivalent similar antitumor efficacy (T/
C ¼ 44.37%) in comparison with the positive control drug Sorafenib
(T/C ¼ 37.35%) without severe body weight reduction (body weight
change: ꢀ0.46 g) (Fig. 5D).
4. Experimental protocols
4.1. Chemistry
All starting materials were obtained from commercial suppliers
and usedwithoutfurtherpurification. NMRspectrawererecorded,on
aBrukerDPX-300spectrometer, inDMSO-d6 orCDCl3-d6 usingTMSas
3. Conclusion
the internal standard. Chemical shifts (d) were reported in parts per
The bis-aryl urea derivative 6a as a novel and potent DFG-out B-
RafV600E inhibitor was evolved from a scaffold combination of UI-
125 and Sorafenib. Subsequent optimization of the ring
Aelinkerering B domain provided a class of bis-aryl amides based
on 2-amino-3-purinylpyridine moiety. All synthesized compounds
were evaluated for B-RafV600E kinase inhibition, as well as mela-
noma A375 antiproliferative potency. The SAR study indicated that
the methyl substitution at C-4 position on the ring B, amides as
linkers between the phenyl rings and lipophilic groups at meta or
para position on ring A are beneficial for improving potency. All
compounds exhibited potent B-RafV600E inhibition with IC50 values
ranging from 0.70 to 13.5 nM, comparing to Vemurafenib
(IC50 ¼ 23.0 nM). Compounds 20cee, 20g and 21h displaying
million downfield from the internal standard. The signals were
quoted as s (singlet), d (doublet), t (triplet), m (multiplet). Analytical
thin-layer chromatography (TLC) was performed with silica gel 60
F254, 0.25 mm per-coated TLC plates. TLC plates were visualized using
UV254 Column chromatography was conducted on silica gel
.
(230e400 mesh). Mass spectra were obtained on a Waters Quattro
Micromass instrument using electrospray ionization (ESI) technique.
4.1.1. General procedure for the preparation of 3-nitrobisaryl ureas
(8a, 8b)
1-Chloro-4-isocyanato-2-(trifluoromethyl)benzene (5 mmol)
was added to a solution of 7a or 7b (5 mmol) in anhydrous

588
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
dichloromethane at room temperature. As the mixture was stirred
for 2 h, pure solid appeared in the solvent. After completion of the
reaction as indicated by TLC, precipitated product was collected by
filtration, washed well with dichloromethane and dried to provide
the intermediates 8a, 8b.
(1H, t, HeAr, J ¼ 8.4 Hz), 7.62 (2H, d, HeAr, J ¼ 1.8 Hz), 7.98 (1H, d,
HeAr, J ¼ 8.4 Hz), 8.27 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.64
(1H, d, HeAr, J ¼ 2.1 Hz), 10.49 (1H, s, NHCO).
4.1.2.5. N-(3-cyanophenyl)-4-methyl-3-nitrobenzamide
(12e).
White solid (0.934 g, 83.0%) was obtaþin1ed from 10b and 11d. Mp:
165e167 ꢂC. ESI-MS m/z: 282.1 [MþH] . H NMR (300 MHz, DMSO-
d6): 2.34 (3H, s, ArCH3), 7.52 (1H, t, HeAr, J ¼ 8.1 Hz), 7.68 (1H, dd,
HeAr, J ¼ 4.8 Hz, J ¼ 8.1 Hz), 7.90 (2H, s, HeAr), 7.70e7.76 (1H, m,
HeAr), 8.32 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.9 Hz), 8.47 (1H, d, HeAr,
J ¼ 2.1 Hz), 10.84 (1H, s, NHCO).
4.1.1.1. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-nitrophenyl)
urea (8a). Light yellow solid (1.58 g, 88.0%) was obtained from 7a.
Mp: 229e231 ꢂC. ESI-MS m/z: 359.0 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): d 7.68e7.88 (5H, m, HeAr), 8.12 (1H, s, HeAr), 8.54 (1H,
d, HeAr, J ¼ 1.8 Hz), 9.38 (1H, s, NHCONH), 9.44 (1H, s, NHCONH).
4.1.1.2. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-
nitrophenyl)urea (8b). Light yellow solid (1.64 g, 87.6%) was ob-
tained from 7b. Mp: 219e220 ꢂC. ESI-MS m/z: 373.0 [MþH]þ. 1H
4.1.2.6. N-(3-fluorophenyl)-4-methyl-3-nitrobenzamide
(12f).
White solid (0.828 g, 75.5%) was obtained from 10b and 11e. Mp:
165e167 ꢂC. ESI-MS m/z: 275.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
d6): 2.49 (3H, s, ArCH3), 6.96e7.02 (1H, m, HeAr), 7.39e7.46 (1H, m,
HeAr), 7.58 (1H, d, HeAr, J ¼ 9.0 Hz), 7.70e7.76 (1H, m, HeAr), 8.01
(1H, d, HeAr, J ¼ 9.0 Hz), 8.26 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz),
8.64 (1H, d, HeAr, J ¼ 2.1 Hz), 10.72 (1H, s, NHCO).
NMR (300 MHz, DMSO-d6): d 2.46 (3H, s, ArCH3), 7.42 (1H, d, HeAr,
J ¼ 8.4 Hz), 7.58e7.69 (3H, m, HeAr), 8.11 (1H, d, HeAr, J ¼ 2.1 Hz),
8.28 (1H, d, HeAr, J ¼ 2.1 Hz), 9.26 (1H, s, NHCONH), 9.31 (1H, s,
NHCONH).
4.1.2. General procedure for the preparation of 3-nitrobenzamide
and N-3-nitrobenzamide analogs (12aeo, 15aeh)
4.1.2.7. N-(4-chloro-3-methylphenyl)-4-methyl-3-nitrobenzamide
(12g). White solid (0.909 g, 74.6%) was obtaiþned from 10b and 11f.
Mp: 208e210 ꢂC. ESI-MS m/z: 305.1 [MþH] . 1H NMR (300 MHz,
DMSO-d6): 2.34 (3H, s, ArCH3), 2.49 (3H, s, ArCH3), 7.42 (1H, d,
HeAr, J ¼ 8.7 Hz), 7.64 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 7.76
(1H, d, HeAr, J ¼ 2.1 Hz), 7.99 (1H, d, HeAr, J ¼ 8.4 Hz), 8.25 (1H, dd,
HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.64 (1H, d, HeAr, J ¼ 2.1 Hz),10.64 (1H,
s, NHCO).
A mixture of 4-substituted-3-nitrobenzoic acid 10aed, 14aeg
(5 mmol), EDCI (6 mmol) and HOBt in anhydrous DMF (20 mL) was
stirred at room temperature for about 0.5e2 h. Then the appro-
priate aniline 11aej, 7aeb (4 mmol) was added with triethylamine
(5 mmol) and stirred at room temperature for 4e24 h. Then the
reaction mixture was diluted to 100 mL with water and extracted
with EtOAc (3 ꢃ 30 mL). The combined organic layers were washed
with saturated solution of NaCl, then dried over MgSO4 and
concentrated under reduced pressure. The residue was chromato-
graphed over silica gel column using acetic ether and petroleum
ether (1:10e1:8, v:v) as eluent to obtain compounds 12aeo, 15aeh
as white solid.
4.1.2.8. N-(3-chlorophenyl)-4-methyl-3-nitrobenzamide
(12h).
White solid (0.902 g, 77.6%) was obtaþin1ed from 10b and 11g. Mp:
142e144 ꢂC. ESI-MS m/z: 291.0 [MþH] . H NMR (300 MHz, DMSO-
d6): 2.47 (3H, s, ArCH3), 7.21 (1H, dd, HeAr, J ¼ 1.8 Hz, J ¼ 7.8 Hz),
7.42 (1H, t, HeAr, J ¼ 8.1 Hz), 7.70 (1H, d, HeAr, J ¼ 8.1 Hz),
7.93e8.08 (2H, m, HeAr), 8.26 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz),
8.64 (1H, d, HeAr, J ¼ 2.1 Hz), 10.69 (1H, s, NHCO).
4.1.2.1. N-(4-chloro-3-(trifluoromethyl)phenyl)-3-nitrobenzamide
(12a). White-off solid (1.03 g, 74.5%) was obtained frþom 10a and
11a. Mp: 189e190 ꢂC. ESI-MS m/z: 345.0 [MþH] . 1H NMR
4.1.2.9. N-(3-bromo-4-chlorophenyl)-4-methyl-3-nitrobenzamide
(12i). White solid (1.24 g, 83.7%) was obtained from 10b and 11h.
Mp: 218e220 ꢂC. ESI-MS m/z: 369.0 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): 2.46 (3H, s, ArCH3), 7.69 (1H, d, HeAr, J ¼ 8.9 Hz), 7.79
(1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.9 Hz), 8.01 (1H, d, HeAr, J ¼ 8.4 Hz),
8.24e8.30 (2H, m, HeAr), 8.64 (1H, d, HeAr, J ¼ 2.1 Hz), 10.76 (1H, s,
NHCO).
(300 MHz, DMSO-d6):
d
7.76 (1H, d, HeAr, J ¼ 8.7 Hz), 7.88 (1H, t,
HeAr, J ¼ 8.1 Hz), 8.13 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.7 Hz), 8.36 (1H,
d, HeAr, J ¼ 2.1 Hz), 8.42 (1H, d, HeAr, J ¼ 2.1 Hz), 8.48 (1H, dd,
HeAr, J ¼ 1.8 Hz, J ¼ 8.1 Hz), 8.83 (1H, t, HeAr, J ¼ 1.8 Hz), 10.96 (1H,
s, CONH).
4.1.2.2. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-
nitrobenzamide (12b). White solid (1.10 g, 76.9%) was obtained
from 10b and 11a. Mp: 174e175 ꢂC. ESI-MS m/z: 359.0 [MþH]þ. 1H
NMR (300 MHz, CDCl3): 2.48 (3H, s, ArCH3), 7.52 (1H, d, HeAr,
J ¼ 8.7 Hz), 7.69 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 9.0 Hz), 8.00 (1H, d,
HeAr, J ¼ 8.7 Hz), 8.24e8.30 (2H, m, HeAr), 8.64 (1H, d, HeAr,
J ¼ 2.1 Hz), 10.83 (1H, s, NHCO).
4.1.2.10. N-(4-(trifluoromethyl)phenyl)-4-methyl-3-nitrobenzamide
(12j). White solid (1.02 g, 78.5%) was obtained from 10b and 11i.
Mp: 145e147 ꢂC. ESI-MS m/z: 325.0 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): 2.46 (3H, s, ArCH3), 7.77 (2H, d, HeAr, J ¼ 8.7 Hz), 8.01
(3H, d, HeAr, J ¼ 8.4 Hz), 8.29 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz),
8.67 (1H, d, HeAr, J ¼ 2.1 Hz), 10.92 (1H, s, NHCO).
4.1.2.3. N-(3-(trifluoromethyl)phenyl)-4-methyl-3-nitrobenzamide
(12c). White solid (0.978 g, 75.4%) was obtained from 10b and 11b.
Mp: 154e156 ꢂC. ESI-MS m/z: 325.0 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): 2.46 (3H, s, ArCH3), 7.53 (1H, d, HeAr, J ¼ 8.4 Hz), 7.82
(1H, t, HeAr, J ¼ 7.8 Hz), 8.02 (2H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz),
8.29 (1H, d, HeAr, J ¼ 7.8 Hz), 8.33 (1H, s, HeAr), 8.54 (1H, d, HeAr,
J ¼ 2.1 Hz), 10.84 (1H, s, NHCO).
4.1.2.11. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-chloro-3-
nitrobenzamide (12k). White solid (1.30 g, 86.1%) was obtained
from 10c and 11a. Mp: 155e157 ꢂC. ESI-MS m/z: 379.0 [MþH]þ. 1H
NMR (300 MHz, DMSO-d6):
d
7.77 (1H, d, HeAr, J ¼ 8.7 Hz), 8.02
(1H, d, HeAr, J ¼ 8.4 Hz), 8.10 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.7 Hz),
8.27 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.32 (1H, d, HeAr,
J ¼ 2.4 Hz), 8.66 (1H, d, HeAr, J ¼ 2.1 Hz), 10.91 (1H, s, NHCO).
4.1.2.4. N-(3-isopropylphenyl)-4-methyl-3-nitrobenzamide
(12d).
4.1.2.12. N-(3-(trifluoromethyl)phenyl)-4-chloro-3-nitrobenzamide
(12l). White solid (1.10 g, 79.7%) was obtained from 10c and 11b.
Mp: 159e160 ꢂC. ESI-MS m/z: 345.0 [MþH]þ. 1H NMR (300 MHz,
Light brown solid (0.893 g, 74.8%) was obtained from 10b and 11c.
Mp: 102e104 ꢂC. ESI-MS m/z: 299.1 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): 1.22 (6H, d, CH(CH3)2, J ¼ 6.9 Hz), 2.48 (3H, s, ArCH3),
2.82e2.93 (1H, m, CH(CH3)2), 7.03 (1H, d, HeAr, J ¼ 7.5 Hz), 7.29
CDCl3):
d
7.51 (1H, d, HeAr, J ¼ 7.8 Hz), 7.64 (1H, t, HeAr, J ¼ 8.1 Hz),
8.01 (1H, d, HeAr, J ¼ 8.1 Hz), 8.05 (1H, d, HeAr, J ¼ 8.1 Hz), 8.20

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
589
(1H, s, HeAr), 8.28 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.67 (1H, d,
HeAr, J ¼ 2.1 Hz), 10.45 (1H, s, NHCO).
4.1.2.21. N-(4-methyl-3-nitrophenyl)-3-chlorobenzamide
(15f).
White solid (0.950 g, 81.6%) was obtained from 14e and 7b. Mp:
121e123 ꢂC. ESI-MS m/z: 291.0 [MþH]þ. 1H NMR (300 MHz, DMSO-
4.1.2.13. N-(3-isopropylphenyl)-4-chloro-3-nitrobenzamide (12m).
White solid (1.10 g, 86.6%) was obtained from 10c and 11c. Mp:
144e145 ꢂC. ESI-MS m/z: 319.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
d6):
d
2.47 (3H, s, ArCH3), 7.50 (1H, d, HeAr, J ¼ 9.0 Hz), 7.69e7.77
(2H, m, HeAr), 7.96 (1H, d, HeAr, J ¼ 7.8 Hz), 8.02 (1H, dd, HeAr,
J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.06 (1H, s, HeAr), 8.56 (1H, d, HeAr,
J ¼ 2.1 Hz), 10.67 (1H, s, NHCO).
d6):
d
1.27 (6H, d, (CH3)2CH, J ¼ 6.9 Hz), 2.89 (1H, m, (CH3)2CH), 6.97
(1H, d, HeAr, J ¼ 7.5 Hz), 7.26 (1H, t, HeAr, J ¼ 7.8 Hz), 7.54e7.59
(2H, m, HeAr), 7.97 (1H, d, HeAr, J ¼ 8.4 Hz), 8.25 (1H, dd, HeAr,
J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.63 (1H, d, HeAr, J ¼ 2.1 Hz), 10.44 (1H, s,
NHCO).
4.1.2.22. N-(4-methyl-3-nitrophenyl)-3-methoxybenzamide
(15g).
White solid (0.948 g, 82.6%) was obtþained from 14f and 7b. Mp:
180e182 ꢂC. ESI-MS m/z: 287.1 [MþH] . 1H NMR (300 MHz, DMSO-
d6):
d 2.47 (3H, s, ArCH3), 3.84 (1H, s, OCH3), 7.19 (1H, dd, HeAr,
4.1.2.14. N-(3,4-dichlorophenyl)-4-chloro-3-nitrobenzamide (12n).
White solid (1.19 g, 81.2%) was obtained from 10c and 11j. Mp:
197e198 ꢂC. ESI-MS m/z: 345.0 [MþH]þ. 1H NMR (300 MHz, DMSO-
J ¼ 1.5 Hz, J ¼ 6.3 Hz), 7.55e7.61 (3H, m, HeAr), 8.00 (1H, dd, HeAr,
J ¼ 2.3 Hz, J ¼ 8.4 Hz), 8.18 (1H, d, HeAr, J ¼ 2.1 Hz), 8.55 (1H, d,
HeAr, J ¼ 2.1 Hz), 10.56 (1H, s, NHCO).
d6):
d
7.68 (1H, d, HeAr, J ¼ 8.7 Hz), 7.76 (1H, dd, HeAr, J ¼ 2.4 Hz,
J ¼ 8.7 Hz), 8.02 (1H, d, HeAr, J ¼ 8.4 Hz), 8.15 (1H, d, HeAr,
J ¼ 2.4 Hz), 8.28 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.66 (1H, d,
HeAr, J ¼ 2.1 Hz), 10.80 (1H, s, NHCO).
4.1.2.23. N-(4-methyl-3-nitrophenyl)-3-(dimethylamino)benzamide
(15h). White solid (0.968 g, 80.7%) was obtaþined from 14g and 7b.
Mp: 107e109 ꢂC. ESI-MS m/z: 300.1 [MþH] . 1H NMR (300 MHz,
DMSO-d6): 2.47 (3H, s, ArCH3), 3.12 (6H, s, N(CH3)2), 7.19e7.22 (2H,
m, HeAr), 7.37e7.45 (2H, m, HeAr), 7.58 (1H, dd, HeAr, J ¼ 2.1 Hz,
J ¼ 8.1 Hz), 7.82e7.84 (2H, m, HeAr), 10.66 (1H, s, NHCO).
4.1.2.15. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-fluoro-3-
nitrobenzamide (12o). White solid (1.19 g, 81.2%) was obtained from
10c and 11j. Mp: 181e182 ꢂC. ESI-MS m/z: 345.0 [MþH]þ. 1H NMR
(300 MHz, DMSO-d6):
d
7.68e7.75 (2H, m, HeAr), 7.92 (1H, d, HeAr,
4.1.3. General procedure for the preparation of 3-aminobisaryl
ureas and amides (9aeb, 13aeo and 16aeh)
J ¼ 2.1 Hz), 8.08 (1H, d, HeAr, J ¼ 8.1 Hz), 8.51e8.56 (2H, m, HeAr),
10.82 (1H, s, NHCO).
To a solution of 3-nitrobisarylurea/3-nitrobenzamide 8aeb,
12aeo and 15aeh (3 mmol) dissolved in methanol/tetrahydrofuran
(1:1, 30 mL) was added 5% palladium/carbon (3 mmol), then the
reaction mixture was stirred at room temperature under hydrogen
atmosphere for 3e6 h. The insoluble material was filtered off
through a short bed of celite, and the filtrate was concentrated in
vacuum. The crude product was purified by column chromatog-
raphy over silica gel using acetic ether and petroleum ether (4:1) as
eluent to give compounds 9aeb, 13aeo and 16aeh as white or light
yellow solid.
4.1.2.16. N-(3-nitrophenyl)-4-chloro-3-(trifluoromethyl)benzamide
(15a). White solid (1.14 g, 82.6%) was obtained from 14a and 7a.
Mp: 205e206 ꢂC. ESI-MS m/z: 345.0 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6):
d
7.70 (1H, t, HeAr, J ¼ 8.1 Hz), 7.96e8.03 (2H, m, HeAr),
8.20 (1H, d, HeAr, J ¼ 8.1 Hz), 8.30 (1H, dd, HeAr, J ¼ 2.1 Hz,
J ¼ 8.4 Hz), 8.45 (1H, d, HeAr, J ¼ 1.8 Hz), 8.78 (1H, t, HeAr,
J ¼ 2.1 Hz), 10.96 (1H, s, NHCO).
4.1.2.17. N-(4-methyl-3-nitrophenyl)-4-chloro-3-(trifluoromethyl)
benzamide (15b). White solid (1.24 g, 86.8%) was obtained from 14a
and 7b. Mp: 189e190 ꢂC. ESI-MS m/z: 359.0 [MþH]þ. 1H NMR
4.1.3.1. 1-(3-Aminophenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)
urea (9a). Light yellow solid (0.629 g, 63.6%)þwas obtained from 8a.
Mp: 210e212 ꢂC. ESI-MS m/z: 330.0 [MþH] . 1H NMR (300 MHz,
(300 MHz, DMSO-d6):
d 2.45 (3H, s, ArCH3), 7.55 (1H, d, HeAr,
J ¼ 8.7 Hz), 7.97e8.03 (2H, m, HeAr), 8.30 (1H, dd, HeAr, J ¼ 2.1 Hz,
J ¼ 8.4 Hz), 8.44 (1H, d, HeAr, J ¼ 1.8 Hz), 8.54 (1H, d, HeAr,
J ¼ 2.1 Hz), 10.86 (1H, s, NHCO).
CDCl3):
d
5.11 (2H, s, ArNH2), 6.21 (1H, dd, HeAr, J ¼ 1.2 Hz,
J ¼ 8.1 Hz), 6.53 (1H, d, HeAr, J ¼ 9.0 Hz), 6.81 (1H, t, HeAr,
J ¼ 2.1 Hz), 6.90 (1H, t, HeAr, J ¼ 7.8 Hz), 7.55e7.62 (2H, m, HeAr),
8.14 (1H, d, HeAr, J ¼ 2.1 Hz), 8.53 (1H, s, NHCONH), 9.02 (1H, s,
NHCONH).
4.1.2.18. N-(4-methyl-3-nitrophenyl)-3-(trifluoromethyl)benzamide
(15c). White solid (1.07 g, 82.6%) was obtained from 14b and 7b.
Mp: 165e167 ꢂC. ESI-MS m/z: 324.0 [MþH]þ. 1H NMR (300 MHz,
4.1.3.2. 1-(3-Amino-4-methylphenyl)-3-(4-chloro-3-(tri-
fluoromethyl)phenyl)urea (9b). White solid (0.720 g, 69.8%)þwas
obtained from 8b. Mp: 199e201 ꢂC. ESI-MS m/z: 344.0 [MþH] . 1H
DMSO-d6):
d
2.45 (3H, s, ArCH3), 7.51 (1H, d, HeAr, J ¼ 7.8 Hz), 7.76
(1H, t, HeAr, J ¼ 7.9 Hz), 8.01 (1H, d, HeAr, J ¼ 8.5 Hz), 8.05 (1H, d,
HeAr, J ¼ 8.4 Hz), 8.21 (1H, s, HeAr), 8.29 (1H, dd, HeAr, J ¼ 2.1 Hz,
J ¼ 8.4 Hz), 8.67 (1H, d, HeAr, J ¼ 2.1 Hz), 10.80 (1H, s, NHCO).
NMR (300 MHz, DMSO-d6):
d 1.98 (3H, s, ArCH3), 4.76 (2H, s,
ArNH2), 6.55 (1H, d, HeAr, J ¼ 8.1 Hz), 6.78 (1H, d, HeAr, J ¼ 8.4 Hz),
6.87 (1H, d, HeAr, J ¼ 2.1 Hz), 7.54e7.63 (2H, m, HeAr), 8.18 (1H, d,
HeAr, J ¼ 2.1 Hz), 9.18 (1H, s, NHCONH), 9.82 (1H, s, NHCONH).
4.1.2.19. N-(4-methyl-3-nitrophenyl)-3-cyanobenzamide
(15d).
White solid (0.934 g, 84.1%) was obtained from 14c and 7b. Mp:
162e163 ꢂC. ESI-MS m/z: 282.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
4.1.3.3. N-(4-chloro-3-(trifluoromethyl)phenyl)-3-aminobenzamide
(13a). White solid (0.665 g, 70.4%) was obtained from 12a. Mp:
168e170 ꢂC. ESI-MS m/z: 315.0 [MþH]þ. 1H NMR (300 MHz, CDCl3):
d6):
d
2.45 (3H, s, ArCH3), 7.57 (1H, dd, HeAr, J ¼ 1.8 Hz, J ¼ 7.8 Hz),
7.81e7.84 (3H, m, HeAr), 8.19 (1H, dd, HeAr, J ¼ 4.8 Hz, J ¼ 9.0 Hz),
8.36 (1H, dd, HeAr, J ¼ 4.5 Hz, J ¼ 8.4 Hz), 8.48 (1H, s, HeAr), 10.79
(1H, s, NHCO).
d
4.27 (2H, s, ArNH2), 6.88e6.93 (2H, m, HeAr), 7.09 (1H, s, HeAr),
7.39 (2H, d, HeAr, J ¼ 2.1 Hz), 7.75 (1H, d, HeAr, J ¼ 2.1 Hz), 7.90 (1H,
s, HeAr), 8.15 (1H, s, CONH).
4.1.2.20. N-(4-methyl-3-nitrophenyl)-4-chloro-3-methylbenzamide
(15e). White solid (0.881 g, 72.2%) was obtained from 14d and 7b.
Mp: 216e217 ꢂC. ESI-MS m/z: 305.1 [MþH]þ. 1H NMR (300 MHz,
DMSO-d6): 2.45 (3H, s, ArCH3), 2.58 (3H, s, ArCH3), 7.57 (2H, dd,
HeAr, J ¼ 1.8 Hz, J ¼ 7.8 Hz), 7.69 (1H, d, HeAr, J ¼ 2.1 Hz), 7.78e7.82
(3H, m, HeAr), 10.79 (1H, s, NHCO).
4.1.3.4. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-
aminobenzamide (13b). White-off solid (0.654 g, 66.3%) was ob-
tained from 12b. Mp: 154e156 ꢂC. ESI-MS m/z: 329.0 [MþH]þ. 1H
NMR (300 MHz, CDCl3):
d 2.24 (3H, s, ArCH3), 3.80 (2H, s, ArNH2),
7.13e7.18 (2H, m, HeAr), 7.21 (1H, s, HeAr), 7.48 (1H, d, HeAr,

590
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
J ¼ 8.7 Hz), 7.88 (1H, d, HeAr, J ¼ 8.7 Hz), 7.94 (1H, s, HeAr), 7.95
NMR (300 MHz, DMSO-d6): d 5.64 (2H, s, ArNH2), 7.12 (1H, d, HeAr,
(1H, s, CONH).
J ¼ 8.1 Hz), 7.34e7.37 (2H, m, HeAr), 7.70 (1H, t, HeAr, J ¼ 8.7 Hz),
8.09 (1H, d, HeAr, J ¼ 8.7 Hz), 8.33 (1H, s, HeAr), 10.54 (1H, s,
CONH).
4.1.3.5. N-(3-(trifluoromethyl)phenyl)-4-methyl-3-aminobenzamide
(13c). White-off solid (0.599 g, 67.2%)þw1as obtained from 12c. Mp:
120e121 ꢂC. ESI-MS m/z: 294.1 [MþH] . H NMR (300 MHz, DMSO-
4.1.3.14. N-(3-trifluoromethylphenyl)-4-chloro-3-aminobenzamide
(13l). White-off solid (0.765 g, 81.0%)þw1as obtained from 12l. Mp:
124e126 ꢂC. ESI-MS m/z: 315.0 [MþH] . H NMR (300 MHz, DMSO-
d6):
d 2.12 (3H, s, ArCH3), 5.09 (2H, s, ArNH2), 7.05e7.12 (2H, m,
HeAr), 7.18 (1H, s, HeAr), 7.41 (1H, d, HeAr, J ¼ 7.8 Hz), 7.57 (1H, t,
HeAr, J ¼ 7.8 Hz), 8.04 (1H, d, HeAr, J ¼ 8.7 Hz), 8.24 (1H, s, HeAr),
10.30 (1H, s, CONH).
d6):
d
5.63 (2H, s, ArNH2), 7.13 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.4 Hz),
7.35 (1H, d, HeAr, J ¼ 2.4 Hz), 7.37 (1H, s, HeAr), 7.44 (1H, d, HeAr,
J ¼ 7.8 Hz), 7.59 (1H, t, HeAr, J ¼ 8.4 Hz), 8.03 (1H, d, HeAr,
J ¼ 8.4 Hz), 8.22 (1H, s, HeAr), 10.44 (1H, s, CONH).
4.1.3.6. N-(3-isopropylphenyl)-4-methyl-3-aminobenzamide (13d).
White-off solid (0.534 g, 66.2%) was obtained from 12d. Mp:
152e154 ꢂC. ESI-MS m/z: 269.1 [MþH]þ. 1H NMR (300 MHz, CDCl3):
4.1.3.15. N-(3-isopropylphenyl)-4-chloro-3-aminobenzamide (13m).
White-off solid (0.587 g, 67.7%) waþs 1obtained from 12m. Mp:
147e148 ꢂC. ESI-MS m/z: 289.1 [MþH] . H NMR (300 MHz, DMSO-
d
1.26 (6H, d, (CH3)2CH, J ¼ 6.9 Hz), 2.20 (3H, s, ArCH3), 2.90e2.93
(1H, m, (CH3)2CH), 3.77 (2H, s, ArNH2), 7.00 (1H, d, HeAr, J ¼ 7.8 Hz),
7.09e7.15 (2H, m, HeAr), 7.22e7.29 (2H, m, HeAr), 7.46 (1H, d,
HeAr, J ¼ 8.1 Hz), 7.50 (1H, s, HeAr), 7.82 (1H, s, CONH).
d6):
d
1.22 (6H, d, CH(CH3)2, J ¼ 6.9 Hz), 2.84e2.93 (1H, m,
CH(CH3)2), 5.15 (2H, s, ArNH2), 6.97 (1H, d, HeAr, J ¼ 7.8 Hz), 7.28
(1H, d, HeAr, J ¼ 7.8 Hz), 7.54e7.59 (2H, m, HeAr), 7.97 (1H, t, HeAr,
J ¼ 8.4 Hz), 8.26 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.63 (1H, d,
HeAr, J ¼ 2.1 Hz), 10.43 (1H, s, CONH).
4.1.3.7. N-(3-cyanophenyl)-4-methyl-3-aminobenzamide
(13e).
White-off solid (0.572 g, 75.7%) was obtained from 12e. Mp:
180e182 ꢂC. ESI-MS m/z: 252.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
d6):
d
2.12 (3H, s, ArCH3), 5.10 (2H, s, ArNH2), 7.08e7.11 (2H, m,
4.1.3.16. N-(3,4-dichlorophenyl)-4-chloro-3-aminobenzamide (13n).
White-off solid (0.691 g, 73.2%) was obtained from 12n. Mp:
182e184 ꢂC. ESI-MS m/z: 315.0 [MþH]þ. 1H NMR (300 MHz, DMSO-
HeAr), 7.17 (1H, s, HeAr), 7.51e7.58 (2H, m, HeAr), 8.02e8.06 (1H,
m, HeAr), 8.24 (1H, s, HeAr), 10.31 (1H, s, CONH).
d6):
d
5.65 (2H, s, ArNH2), 7.10 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.1 Hz),
4.1.3.8. N-(3-fluorophenyl)-4-methyl-3-aminobenzamide
(13f).
7.32e7.37 (2H, m, HeAr), 7.61 (1H, d, HeAr, J ¼ 8.7 Hz), 7.73 (1H, dd,
HeAr, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 8.13 (1H, d, HeAr, J ¼ 2.4 Hz),10.42 (1H,
s, CONH).
White-off solid (0.525 g, 71.4%) was obtained from 12f. Mp:
142e144 ꢂC. ESI-MS m/z: 245.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
d6):
d 2.42 (3H, s, ArCH3), 5.08 (2H, s, ArNH2), 6.85e6.92 (1H, m,
HeAr), 7.06 (1H, d, HeAr, J ¼ 8.7 Hz), 7.17 (2H, d, HeAr, J ¼ 4.8 Hz),
7.35 (1H, dd, HeAr, J ¼ 4.8 Hz, J ¼ 8.7 Hz), 7.56 (1H, d, HeAr,
J ¼ 8.7 Hz), 7.74 (1H, t, HeAr, J ¼ 4.8 Hz), 10.18 (1H, s, CONH).
4.1.3.17. N-(4-chloro-3-(trifluoromethyl)phenyl)-4-fluoro-3-
aminobenzamide (13o). White-off solid (0.722 g, 72.3%) was ob-
tained from 12o. Mp: 174e175 ꢂC. ESI-MS m/z: 333.0 [MþH]þ. 1H
NMR (300 MHz, DMSO-d6): d 5.43 (2H, s, ArNH2), 7.15 (2H, d, HeAr,
4.1.3.9. N-(4-chloro-3-methylphenyl)-4-methyl-3-aminobenzamide
(13g). White-off solid (0.572 g, 69.3%)þw1as obtained from 12g. Mp:
156e157 ꢂC. ESI-MS m/z: 275.0 [MþH] . H NMR (300 MHz, CDCl3):
J ¼ 8.7 Hz), 7.35 (1H, d, HeAr, J ¼ 9.0 Hz), 7.70 (1H, d, HeAr,
J ¼ 9.0 Hz), 8.09 (1H, dd, HeAr, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 8.34 (1H, d,
HeAr, J ¼ 2.4 Hz), 10.49 (1H, s, CONH).
d
2.25 (3H, s, ArCH3), 2.39 (3H, s, ArCH3), 5.11 (2H, s, ArNH2), 7.14
(2H, s, HeAr), 7.31 (2H, d, HeAr), 7.41 (1H, dd, HeAr, J ¼ 2.1 Hz,
4.1.3.18. N-(3-aminophenyl)-4-chloro-3-(trifluoromethyl)benzamide
(16a). White-off solid (0.801 g, 84.8%)þw1as obtained from 15a. Mp:
194e195 ꢂC. ESI-MS m/z: 315.0 [MþH] . H NMR (300 MHz, DMSO-
J ¼ 8.9 Hz), 7.57 (1H, d, HeAr, J ¼ 2.1 Hz), 7.80 (1H, s, CONH).
4.1.3.10. N-(3-chlorophenyl)-4-methyl-3-aminobenzamide
(13h).
d6):
d
5.67 (2H, s, ArNH2), 7.03 (1H, dd, HeAr, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
White-off solid (0.579 g, 74.0%) was obtained from 12h. Mp:
7.32 (1H, t, HeAr, J ¼ 7.8 Hz), 7.46 (1H, d, HeAr, J ¼ 7.8 Hz), 7.56 (1H,
d, HeAr, J ¼ 7.8 Hz), 7.92 (1H, d, HeAr, J ¼ 8.4 Hz), 8.28 (1H, d, HeAr,
J ¼ 2.1 Hz, J ¼ 8.4 Hz), 8.37 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 4.8 Hz),
10.51 (1H, s, CONH).
136e138 ꢂC. ESI-MS m/z: 261.0 [MþH]þ. 1H NMR (300 MHz, DMSO-
d6):
d 2.12 (3H, s, ArCH3), 5.08 (2H, s, ArNH2), 7.06e7.16 (4H, m,
HeAr), 7.35 (1H, t, HeAr, J ¼ 8.1 Hz), 7.69 (1H, d, HeAr, J ¼ 8.1 Hz),
7.96 (1H, s, HeAr), 10.15 (1H, s, CONH).
4.1.3.19. N-(3-amino-4-methylphenyl)-4-chloro-3-(trifluoromethyl)
benzamide (16b). White-off solid (0.836 g, 84.7%) waþs obtained
from 15a. Mp: 180e182 ꢂC. ESI-MS m/z: 329.0 [MþH] . 1H NMR
4.1.3.11. N-(3-bromo-4-chlorophenyl)-4-methyl-3-aminobenzamide
(13i). White-off solid (0.767 g, 75.4%)þw1as obtained from 12i. Mp:
188e190 ꢂC. ESI-MS m/z: 339.0 [MþH] . H NMR (300 MHz, DMSO-
(300 MHz, DMSO-d6): d 2.03 (3H, s, ArCH3), 4.91 (2H, s, ArNH2), 6.82
d6):
d
2.12 (3H, s, ArCH3), 5.10 (2H, s, ArNH2), 7.06 (2H, s, HeAr), 7.15
(1H, d, HeAr, J ¼ 8.1 Hz), 6.88 (1H, d, HeAr, J ¼ 8.1 Hz), 7.08 (1H, s,
HeAr), 7.89 (1H, d, HeAr, J ¼ 8.7 Hz), 8.23 (1H, d, HeAr, J ¼ 8.7 Hz),
8.34 (1H, s, HeAr), 10.18 (1H, s, CONH).
(1H, s, HeAr), 7.58 (1H, d, HeAr, J ¼ 8.7 Hz), 7.79 (1H, dd, HeAr,
J ¼ 8.7 Hz), 8.28 (1H, s, HeAr), 10.23 (1H, s, CONH).
4.1.3.12. N-(4-trifluoromethylphenyl)-4-methyl-3-aminobenzamide
(13j). White-off solid (0.657 g, 74.2%)þw1as obtained from 12j. Mp:
125e127 ꢂC. ESI-MS m/z: 295.0 [MþH] . H NMR (300 MHz, DMSO-
4.1.3.20. N-(3-amino-4-methylphenyl)-3-(trifluoromethyl)benza-
mide (16c). White-off solid (0.736 g, 83.4%) was obtained from 15c.
Mp: 157e159 ꢂC. ESI-MS m/z: 294.1 [MþH]þ. 1H NMR (300 MHz,
d6):
d
2.12 (3H, s, ArCH3), 5.10 (2H, s, ArNH2), 7.05e7.15 (2H, m,
DMSO-d6): d 2.03 (3H, s, ArCH3), 4.87 (2H, s, ArNH2), 6.80e6.89 (2H,
HeAr), 7.18 (1H, s, HeAr), 7.69 (2H, d, HeAr, J ¼ 8.7 Hz), 8.00 (2H, d,
m, HeAr), 7.11 (1H, d, HeAr, J ¼ 2.1 Hz), 7.76 (1H, t, HeAr, J ¼ 7.8 Hz),
7.94 (1H, d, HeAr, J ¼ 7.8 Hz), 8.21e8.25 (2H, m, HeAr), 10.13 (1H, s,
CONH).
HeAr, J ¼ 8.7 Hz), 10.34 (1H, s, CONH).
4.1.3.13. N-(4-chloro-3-trifluoromethylphenyl)-4-chloro-3-
aminobenzamide (13k). White-off solid (0.752 g, 71.8%) was ob-
tained from 12k. Mp: 136e138 ꢂC. ESI-MS m/z: 349.0 [MþH]þ. 1H
4.1.3.21. N-(3-amino-4-methylphenyl)-3-cyanobenzamide
White-off solid (0.624 g, 82.5%) was obtained from 15d. Mp:
(16d).

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
591
166e167 ꢂC. ESI-MS m/z: 252.1 [MþH]þ. 1H NMR (300 MHz, DMSO-
was obtained by a column chromatography, using a mixture of
acetic ether and petroleum ether (1:1) purified as eluþent (1.54 g,
51.5%). Mp: 132e133 ꢂC. ESI-MS m/z: 300.2 [MþH] . 1H NMR
d6):
d 2.44 (3H, s, ArCH3), 5.42 (2H, s, ArNH2), 6.98 (1H, dd, HeAr,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.25 (1H, d, HeAr, J ¼ 8.4 Hz), 7.38 (1H, dd,
HeAr, J ¼ 2.1 Hz, J ¼ 8.1 Hz), 7.74 (1H, t, HeAr, J ¼ 7.8 Hz), 8.04e8.07
(1H, m, HeAr), 8.27 (1H, d, HeAr, J ¼ 8.1 Hz), 8.36 (1H, dd, HeAr,
J ¼ 2.1 Hz, J ¼ 4.8 Hz), 10.33 (1H, s, CONH).
(300 MHz, DMSO-d6): d 1.60e1.67 (2H, m, H-pyran), 1.75e1.82 (1H,
m, H-pyran), 1.99e2.08 (2H, m, H-pyran), 2.34e2.41 (1H, m, H-
pyran), 3.71e3.80 (1H, m, H-pyran), 4.05 (1H, d, J ¼ 11.1 Hz, H-
pyran), 5.86 (1H, dd, J ¼ 2.1 Hz, J ¼ 10.8 Hz, H-pyran), 7.59e7.64 (1H,
m, H-pyridine), 8.47 (1H, dd, H-pyridine, J ¼ 1.0 Hz, J ¼ 3.0 Hz),
8.54e8.60 (1H, m, H-pyridine), 8.90 (1H, s, H-purine), 9.09 (1H, s,
H-purine).
4.1.3.22. N-(3-amino-4-methylphenyl)-4-chloro-3-methylbenzamide
(16e). White-off solid (0.668 g, 81.0%)þwas obtained from 15e. Mp:
176e178 ꢂC. ESI-MS m/z: 275.1 [MþH] . 1H NMR (300 MHz, DMSO-
d6):
d 2.21 (3H, s, ArCH3), 2.34 (3H, s, ArCH3), 5.87 (2H, s, ArNH2),
6.83 (1H, s, HeAr), 6.92e6.96 (2H, m, HeAr), 7.57 (1H, d, HeAr,
J ¼ 7.8 Hz), 7.95 (1H, d, HeAr, J ¼ 7.8 Hz), 8.17 (1H, dd, HeAr,
J ¼ 2.1 Hz, J ¼ 4.8 Hz), 10.15 (1H, s, CONH).
4.1.6. General procedure for the preparation of target compounds
(6aeb, 20aeo and 21aeh)
A mixture of 9aeb, 13aeo or 16aeh (0.55 mmol) and the in-
termediate 20 (165 mg, 0.5 mmol) in anhydrous tetrahydrofuran
(30 mL) was stirred under nitrogen at 0 ꢂC for 10 min. Then LiHMDS
(Lithium hexamethyldisilazide, 1 mol/L THF, 0.5 mL) was added
dropwise to the reaction mixture at 0 ꢂC. Reaction mixture was
further stirred for 2e6 h at room temperature. The mixture was
quenched with H2O (40 mL) and EtOAc (40 mL). The aqueous phase
was washed with EtOAc (3 ꢃ 40 mL) and dried over anhydrous
Na2SO4. After filtration, the solvent was removed under reduced
pressure. Afterward, the residue was dissolved in MeOH (30 mL)
and stirred at room temperature. Then 2 mL diluted hydrochloric
acid (1 M) was added dropwise to the reaction mixture. After
stirring for 3 h at room temperature, adjust the pH to 8-9 by 10%
sodium carbonate solution. The solvent was evaporated under
reduced pressure. The crude compound was purified by column
chromatography over silica gel by using petroleum/ether acetate
(1:1e1:2) as eluent to give compounds 6aeb, 20aeo and 21aeh as
yellow solid.
4.1.3.23. N-(3-amino-4-methylphenyl)-3-chlorobenzamide
(16f).
White-off solid (0.621 g, 79.3%) was obtained from 15f. Mp:
132e134 ꢂC. ESI-MS m/z: 261.0 [MþH]þ. 1H NMR (300 MHz, CDCl3):
d
2.46 (3H, s, ArCH3), 5.21 (2H, s, ArNH2), 7.02e7.06 (1H, m, HeAr),
7.24 (1H, d, HeAr, J ¼ 8.7 Hz), 7.47 (1H, d, HeAr, J ¼ 8.1 Hz), 7.57 (1H,
t, HeAr, J ¼ 7.8 Hz), 7.66 (1H, d, HeAr, J ¼ 8.7 Hz), 7.94 (1H, d, HeAr,
J ¼ 7.8 Hz), 8.36 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 10.30 (1H, s,
CONH).
4.1.3.24. N-(3-amino-4-methylphenyl)-3-methoxybenzamide (16g).
White-off solid (0.617 g, 80.1%) was obtained from 15g. Mp:
116e117 ꢂC. ESI-MS m/z: 257.1 [MþH]þ. 1H NMR (300 MHz, CDCl3):
d
2.15 (3H, s, ArCH3), 3.65 (2H, s, ArNH2), 3.87 (3H, s, OCH3), 6.72
(1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 7.8 Hz), 7.01 (1H, d, HeAr, J ¼ 8.1 Hz),
7.04e7.09 (1H, m, HeAr), 7.29 (1H, d, HeAr, J ¼ 2.1 Hz), 7.38 (2H, d,
HeAr, J ¼ 8.1 Hz), 7.41e7.43 (1H, m, HeAr), 7.67 (1H, s, CONH).
4.1.3.25. N-(3-amino-4-methylphenyl)-3-(dimethylamino)benza-
mide (16h). White-off solid (0.658 g, 81.3%) was obtained from 15h.
Mp: 170e172 ꢂC. ESI-MS m/z: 270.1 [MþH]þ. 1H NMR (300 MHz,
4.1.6.1. 1-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (6a). Yellow solid (9þ1 mg,
34.7%) was obtained from 9a. Mp: 270e272 ꢂC. HRMS (ESI ) m/z
calculated for C24H16ClF3N8O [MþH]þ 525.1160; found, 525.1164. 1H
CDCl3):
d 2.42 (3H, s, ArCH3), 2.97 (6H, s, N(CH3)2), 4.74 (2H, s,
ArNH2), 6.92 (1H, dd, HeAr, J ¼ 1.6 Hz, J ¼ 8.1 Hz), 7.02 (1H, dd,
HeAr, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.23e7.25 (2H, m, HeAr), 7.31 (1H, t,
HeAr, J ¼ 8.1 Hz), 7.43 (1H, dd, HeAr, J ¼ 2.1 Hz, J ¼ 8.1 Hz), 8.34 (1H,
dd, HeAr, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 10.10 (1H, s, CONH).
NMR (300 MHz, DMSO-d6):
d
7.00 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
7.21e7.26 (2H, m), 7.36 (1H, d, J ¼ 6.6 Hz), 7.56e7.66 (2H, m, HeAr),
8.01 (1H, s, HeAr), 8.10 (1H, d, J ¼ 1.8 Hz), 8.34 (1H, dd, J ¼ 1.8 Hz,
J ¼ 4.8 Hz), 8.68 (1H, s), 8.86 (1H, s, H-purine), 9.07 (1H, s, H-pu-
rine), 9.14 (1H, s, NHCONH), 9.88 (1H, s, NHCONH), 12.52 (1H, s),
4.1.4. 6-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (18)
A mixture of 6-chloro-9H-purine 17 (2.01 g, 13 mmol), 3,4-
dihydro-2H-pyran (3.30 g, 39 mmol) and TsOH (516 mg,
0.3 mmol) dissolved in anhydrous ethyl acetate (30 mL) was
refluxed for 3 h. Then cooled to the room temperature and washed
with water and brine. The organic layer was dried (anhydrous
Na2SO4) and concentrated to afford the compound 18 (2.62 g,
83.2%) as a yellow oil. ESI-MS m/z: 239.0 [MþH]þ. 1H NMR
13.81 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 110.20,
112.28, 112.88, 113.85, 114.42, 116.57, 120.97, 122.17, 122.87, 124.58,
126.62 (q, CF3), 128.86, 129.33, 131.86, 139.35, 139.49, 140.87, 141.67,
144.62, 149.74, 150.04, 152.33, 153.03, 153.93 (NHCONH).
4.1.6.2. 1-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea
(6b).
Yellow solid (88 mg, 32.7%) was obtained from 9b. Mp: 246e248 ꢂC.
HRMS (ESIþ) m/z calculated for C25H18ClF3N8O [MþH]þ 539.1317;
(300 MHz, DMSO-d6): d 1.58e1.64 (2H, m, H-pyran), 1.69e1.78 (1H,
m, H-pyran), 1.97e2.05 (2H, m, H-pyran), 2.32e2.37 (1H, m, H-
pyran), 3.69e3.77 (1H, m, H-pyran), 4.03 (1H, d, J ¼ 5.4 Hz, H-py-
ran), 5.80 (1H, dd, J ¼ 2.1 Hz, J ¼ 10.9 Hz, H-pyran), 8.81 (1H, s, H-
purine), 8.91 (1H, s, H-purine).
found, 539.1316. 1H NMR (300 MHz, DMSO-d6):
d 2.42 (3H, s,
ArCH3), 7.05 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.17 (1H, d, J ¼ 8.4 Hz),
7.28 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.1 Hz), 7.61 (2H, t, J ¼ 4.8 Hz), 8.10 (1H,
s), 8.35 (1H, dd, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.39 (1H, d, J ¼ 1.8 Hz), 8.64
(1H, s), 8.73 (1H, s, H-purine), 8.97 (1H, s, H-purine), 9.06 (1H, s,
NHCONH), 9.75 (1H, s, NHCONH), 12.33 (1H, s), 13.78 (1H, s, 9H-
4.1.5. 6-(2-Fluoropyridin-3-yl)-9-(tetrahydro-2H-pyran-2-yl)-9H-
purine (19)
purine). 13C NMR (75 MHz, DMSO-d6):
d 19.17 (CH3), 111.24, 112.42,
A mixture of compound 18 (2.38 g, 10 mmol), (2-fluoropyridin-
3-yl)boronic acid (2.10 g, 15 mmol), PdCl2(dppf) (150 mg, 0.2 mmol)
and Na2CO3 (4.23 g, 40 mmol) dissolved in 1,4-dioxane/H2O (1:7)
(40 mL) was refluxed under nitrogen for 14 h. After cooling to room
temperature, the mixture was diluted in 50 mL H2O. The aqueous
layer was extracted with EtOAc (3 ꢃ 50 mL). The combined organic
layers were washed with saturated solution of NaCl and then dried
over anhydrous MgSO4, filtered and evaporated. The resulting solid
113.11, 113.62, 116.45, 120.96, 121.58, 121.98, 122.67, 124.58, 126.63
(q, CF3), 129.33, 130.06, 131.78, 137.11, 139.21, 139.44, 141.71, 144.46,
149.63, 149.74, 152.38, 153.01, 154.11 (NHCONH).
4.1.6.3. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(4-chloro-3-
(trifluoromethyl)phenyl)benzamide (20a). Yellow solid (74 mg,
29.1%) was obtained from 13a. Mp: 334e335 ꢂC. HRMS (ESIþ) m/z
calculated for C24H15ClF3N7O [MþH]þ 510.1051; found, 510.1048. 1H

592
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
NMR (300 MHz, DMSO-d6):
d
7.06 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
(1H, s, H-purine), 9.10 (1H, s, H-purine), 9.84 (1H, d, J ¼ 6.9 Hz),
10.47 (1H, s, NHCO), 12.46 (1H, s), 13.86 (1H, s, 9H-purine). 13C NMR
7.51 (1H, t, J ¼ 8.1 Hz), 7.59 (1H, d, J ¼ 7.8 Hz), 7.73 (1H, d, J ¼ 8.7 Hz),
8.14e8.18 (2H, m), 8.36e8.38 (2H, m), 8.45 (1H, d, J ¼ 2.4 Hz), 8.56
(1H, s, H-purine), 9.10 (1H, s, H-purine), 9.81 (1H, d, J ¼ 6.7 Hz),
10.96 (1H, s, CONH), 12.98 (1H, s). 13C NMR (75 MHz, DMSO-d6):
(75 MHz, DMSO-d6):
d 18.77 (CH3), 111.35, 113.18, 114.09, 118.71
(CN), 120.39, 121.10, 122.89, 124.72, 126.84, 129.33, 129.98, 132.24,
132.37, 139.20, 140.11, 141.70, 144.76, 149.93, 151.93, 152.97, 153.31,
154.07, 165.37, 166.12 (NHCO). IR (KBr, cmꢀ1): 3431, 3084, 2986,
2822, 1699, 1578, 1483, 858, 791, 762.
d
113.89,114.28,119.11,120.76,121.70,123.13,123.88,124.09,125.08,
126.38 (q, CF3), 128.66, 130.18, 131.80, 134.73, 138.87, 140.87, 141.56,
147.55, 149.25, 149.40, 150.69, 153.80, 155.48, 166.16 (NHCO).
4.1.6.8. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(3-
fluorophenyl)-4-methylbenzamide (20f). Yellow solid (64 mg, 30.1%)
was obtained from 13f. Mp: 315e317 ꢂC. HRMS (ESIþ) m/z calcu-
lated for C24H18FN7O [MþH]þ 440.1630; found, 440.1626 1H NMR
4.1.6.4. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(4-chloro-3-
(trifluoromethyl)phenyl)-4-methylbenzamide (20b). Yellow solid
(71 mg, 27.5%) was obtained from 13b. Mp: 315e317 ꢂC. HRMS
(ESIþ) m/z calculated for C25H17ClF3N7O [MþH]þ 524.1208; found,
[300 MHz, DMSO-d6]: d 2.54 (3H, s, ArCH3), 6.88e6.94 (1H, m), 7.06
524.1213. 1H NMR [300 MHz, DMSO-d6]:
d
2.54 (3H, s, ArCH3), 7.07
(1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.34e7.44 (2H, m) 7.55e7.59 (2H, m),
7.74e7.80 (1H, m), 8.37 (1H, dd, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.73 (1H, s),
8.91 (1H, s, H-purine), 9.10 (1H, s, H-purine), 9.83 (1H, s), 10.35 (1H,
s, CONH), 12.43 (1H, s), 13.85 (1H, s, 9H-purine). 13C NMR (75 MHz,
(1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.45 (1H, d, J ¼ 8.7 Hz), 7.59 (1H, d,
J ¼ 8.7 Hz), 7.71 (1H, d, J ¼ 8.7 Hz), 8.13 (1H, d, J ¼ 8.7 Hz), 8.37 (1H,
d, J ¼ 1.8 Hz), 8.38e8.40 (1H, m), 8.73 (1H, s), 8.95 (1H, s, H-purine),
9.10 (1H, s, H-purine), 9.83 (1H, s),10.56 (1H, s, CONH),12.45 (1H, s),
DMSO-d6):
d 18.63 (CH3), 106.70, 107.04, 109.97, 113.20, 114.06,
13.86 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d
18.79
115.87 (d, J ¼ 2.6 Hz), 120.44, 121.10, 129.36, 130.01 (d, J ¼ 4.5 Hz),
130.14, 132.75, 139.18, 141.11, 141.72, 144.82, 149.92, 151.93, 153.05,
154.12, 160.43, 163.63, 166.07 (NHCO). IR (KBr, cmꢀ1): 3433, 3098,
2990, 2847, 1674, 1599, 1570, 1530, 860, 760.
(CH3), 113.21, 114.11, 118.86 (q, CF3), 120.44, 120.96, 121.11, 123.98,
124.57, 124.79, 126.33, 126.74, 129.32, 130.01, 131.84, 132.24, 132.38,
138.83, 139.25, 141.73, 144.71, 149.91, 149.96, 154.10, 166.16 (CONH).
IR (KBr, cmꢀ1): 3186, 3092, 2989, 2837, 1674, 1593, 1541, 1381, 924,
761.
4.1.6.9. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(4-chloro-3-
methylphenyl)-4-methylbenzamide (20g). Yellow solid (83 mg,
36.6%) was obtained from 13g. Mp: 303e305 ꢂC. HRMS (ESIþ) m/z
calculated for C25H20ClN7O [MþH]þ 470.1491; found, 470.1489. 1H
4.1.6.5. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-methyl-N-(3-
(trifluoromethyl)phenyl)benzamide (20c). Yellow solid (80 mg,
33.4%) was obtained from 13c. Mp: 288e290 ꢂC. HRMS (ESIþ) m/z
calculated for C25H18F3N7O [MþH]þ 490.1598; found, 490.1602. 1H
NMR [300 MHz, DMSO-d6]:
d 2.33 (3H, s, ArCH3), 2.53 (3H, s,
ArCH3), 7.06 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.38 (1H, d, J ¼ 8.7 Hz),
7.42 (1H, d, J ¼ 8.7 Hz), 7.58 (1H, d, J ¼ 7.8 Hz) 7.65 (1H, dd,
J ¼ 2.4 Hz, J ¼ 4.8 Hz), 7.81 (1H, d, J ¼ 2.4 Hz), 8.37 (1H, dd, J ¼ 1.8 Hz,
J ¼ 4.8 Hz), 8.73 (1H, s), 8.90 (1H, s, H-purine), 9.10 (1H, s, H-pu-
rine), 9.82 (1H, s), 10.21 (1H, s, NHCO), 12.42 (1H, s), 13.86 (1H, s,
NMR [300 MHz, DMSO-d6]:
d 2.54 (3H, s, ArCH3), 7.07 (1H, dd,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.44 (2H, d, J ¼ 8.1 Hz), 7.60 (2H, t, J ¼ 7.8 Hz),
8.07 (1H, d, J ¼ 8.7 Hz), 8.28 (1H, s), 8.37 (1H, dd, J ¼ 4.8 Hz,
J ¼ 7.8 Hz), 8.73 (1H, s), 8.94 (1H, s, H-purine), 9.5 (1H, s, H-purine),
9.82 (1H, s), 10.47 (1H, s, NHCO), 12.44 (1H, s), 13.85 (1H, s, 9H-
9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 18.76 (CH3), 19.82
purine). 13C NMR (75 MHz, DMSO-d6):
d
18.77 (CH3), 113.18, 114.02,
(CH3), 113.16, 114.02, 119.39, 120.48, 121.06, 122.65, 127.31, 128.74,
129.44, 129.91, 131.94, 132.85, 135.29, 138.21, 139.14, 141.72, 144.64,
149.90, 149.94, 151.99, 152.98, 154.13, 165.80 (NHCO). IR (KBr,
cmꢀ1): 3431, 3341, 3092, 2972, 2824, 1657, 1572, 1535, 827, 764.
116.27 (d, J ¼ 4.1 Hz), 119.64 (d, J ¼ 3.6 Hz), 120.44, 121.07, 122.34,
123.67,125.96,128.67, 129.08, 129.63 (q, CF3), 129.95, 132.16,132.52,
139.22, 140.12, 141.71, 144.69, 149.88, 149.91, 151.95, 154.11, 166.17
(CONH). IR (KBr, cmꢀ1): 3428, 3088, 2988, 2826, 1670, 1576, 1483,
799, 758, 706.
4.1.6.10. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(3-
chlorophenyl)-4-methylbenzamide (20h). Yellow solid (77 mg,
34.3%) was obtained from 13h. Mp: 278e280 ꢂC. HRMS (ESIþ) m/z
calculated for C24H18ClN7O [MþH]þ 456.1334; found, 456.1331. 1H
4.1.6.6. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(3-
isopropylphenyl)-4-methylbenzamide (20d). Yellow solid (6þ5 mg,
28.1%) was obtained from 13c. Mp: 156e158 ꢂC. HRMS (ESI ) m/z
calculated for C27H25N7O [MþH]þ 464.2193; found, 464.2192. 1H
NMR [300 MHz, DMSO-d6]:
d 2.53 (3H, s, ArCH3), 7.07 (1H, dd,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.15 (1H, d, J ¼ 8.7 Hz), 7.35e7.44 (2H, m), 7.58
(1H, d, J ¼ 8.7 Hz), 7.73 (1H, d, J ¼ 8.7 Hz), 7.99 (1H, s), 8.37 (1H, d,
J ¼ 4.8 Hz), 8.73 (1H, s), 8.91 (1H, s, H-purine), 9.10 (1H, s, H-purine),
9.83 (1H, s), 10.32 (1H, s, CONH), 12.43 (1H, s), 13.85 (1H, s, 9H-
NMR [300 MHz, DMSO-d6]:
d
1.22 (6H, d, (CH3)2CH, J ¼ 6.9 Hz), 2.53
(3H, s, ArCH3), 2.87e2.89 (1H, m, (CH3)2CH), 6.97 (1H, d, J ¼ 7.8 Hz),
7.05 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.25 (1H, t, J ¼ 8.7 Hz), 7.41 (1H,
d, J ¼ 8.7 Hz), 7.57e7.64 (2H, m), 7.68 (1H, s), 8.37 (1H, dd, J ¼ 1.8 Hz,
J ¼ 4.8 Hz), 8.72 (1H, s), 8.87 (1H, s, H-purine), 9.10 (1H, s, H-pu-
rine), 9.81 (1H, s), 10.07 (1H, s, NHCO), 12.39 (1H, s), 13.85 (1H, s,
purine). 13C NMR (75 MHz, DMSO-d6):
d 17.68 (CH3), 111.73, 113.58,
117.48, 119.77, 120.36, 122.03, 127.94, 128.44, 129.96, 130.30, 132.06,
132.60, 134.51, 134.87, 138.94, 142.19, 144.56, 144.85, 146.47, 148.02,
152.43, 153.88, 164.78 (NHCO). IR (KBr, cmꢀ1): 3430, 3088, 2988,
2834, 1667, 1595, 1526, 858, 768.
9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 18.70 (CH3), 23.81
(CH(CH3)2), 33.50 (CH(CH3)2), 113.16, 113.96, 117.94, 118.31, 120.63,
121.10, 121.51, 128.35, 129.37, 129.87, 131.84, 133.22, 139.13, 139.31,
141.71, 144.63, 148.74, 149.91, 149.92, 152.04, 152.92, 154.21, 165.76
(CONH). IR (KBr, cmꢀ1): 3082, 2959, 2918, 2849, 1626, 1587, 1568,
1530, 926, 768.
4.1.6.11. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(3-bromo-4-
chlorophenyl)-4-methylbenzamide (20i). Yellow solid (65 mg,
30.0%) was obtained from 13i. Mp: 335e336 ꢂC. HRMS (ESIþ) m/z
calculated for C24H17BrClN7O [MþH]þ 534.0439; found, 534.0435.
4.1.6.7. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(3-
cyanophenyl)-4-methylbenzamide (20e). Yellow solid (82 mg, 41.0%)
was obtained from 13e. Mp: 328e329 ꢂC. HRMS (ESIþ) m/z calcu-
lated for C25H18N8O [MþH]þ 447.1676; found 447.1674. 1H NMR
1H NMR [300 MHz, DMSO-d6]:
d 2.53 (3H, s, ArCH3), 7.06 (1H, dd,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.43 (1H, d, J ¼ 8.7 Hz), 7.56e7.61 (2H, m), 7.82
(1H, dd, J ¼ 2.4 Hz, J ¼ 8.7 Hz), 8.32 (1H, d, J ¼ 2.4 Hz), 8.37 (1H, dd,
J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.73 (1H, s, H-purine), 8.93 (1H, d, J ¼ 1.8 Hz),
9.10 (1H, s, H-purine), 9.83 (1H, s),10.40 (1H, s, CONH),12.44 (1H, s),
[300 MHz, DMSO-d6]:
d
2.49 (3H, s, ArCH3), 7.07 (1H, dd, J ¼ 4.8 Hz,
J ¼ 7.8 Hz), 7.44 (1H, d, J ¼ 7.8 Hz), 7.54e7.61 (3H, m), 8.05e8.09
13.85 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d
18.79
(1H, m), 8.28 (1H, d, J ¼ 1.2 Hz), 8.36e8.38 (1H, m), 8.73 (1H, s), 8.94
(CH3), 113.19, 114.09, 120.41, 120.66, 121.03, 121.08, 124.46, 126.83,

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
593
129.43, 129.98, 130.21, 132.24, 132.40, 139.21, 139.42, 141.72, 144.71,
149.90,149.95,151.96,153.02, 154.10,166.02 (NHCO). IR (KBr, cmꢀ1):
3343, 3088, 2969, 2828, 1659, 1580, 1528, 866, 800, 762.
153.02, 153.30, 165.11 (NHCO). IR (KBr, cmꢀ1): 3192, 3088, 2963,
2868, 2835, 1653, 1603, 1576, 1526, 762, 702.
4.1.6.16. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-chloro-N-(3,4-
dichlorophenyl)benzamide (20n). Yellow solid (76 mg, 32.4%) was
obtained from 13n. Mp: 322e323 ꢂC. HRMS (ESIþ) m/z calculated
for C23H14Cl3N7O [MþH]þ 510.0398; found, 510.0412. 1H NMR
4.1.6.12. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-methyl-N-(4-
(trifluoromethyl)phenyl)benzamide (20j). Yellow solid (91 mg,
37.8%) was obtained from 13j. Mp: 304e306 ꢂC. HRMS (ESIþ) m/z
calculated for C25H18F3N7O [MþH]þ 490.1598; found, 490.1592. 1H
[300 MHz, DMSO-d6]:
d
7.18 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
7.56e7.64 (2H, m), 7.70e7.79 (2H, m), 8.18 (1H, d, J ¼ 2.4 Hz), 8.46
(1H, dd, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.75 (1H, s, H-purine), 9.13 (1H, s, H-
purine), 9.37 (1H, d, J ¼ 1.8 Hz), 9.85 (1H, s), 10.56 (1H, s, CONH),
13.18 (1H, s), 13.89 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
NMR [300 MHz, DMSO-d6]:
d 2.54 (3H, s, ArCH3), 7.07 (1H, dd,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.44 (1H, d, J ¼ 8.4 Hz), 7.60 (1H, dd, J ¼ 1.8 Hz,
J ¼ 7.8 Hz), 7.72 (2H, d, J ¼ 8.7 Hz), 8.04 (2H, d, J ¼ 8.4 Hz), 8.37 (1H,
dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.73 (1H, s), 8.94 (2H, d, J ¼ 1.5 Hz), 9.11
(1H, s, H-purine), 10.50 (1H, s, CONH), 12.45 (1H, s),13.86 (1H, s, 9H-
d
114.20, 115.23, 120.20, 120.38, 120.94, 121.40, 125.10, 125.81,
purine). 13C NMR (75 MHz, DMSO-d6):
d 17.68 (CH3), 107.90, 111.58,
129.07, 129.81, 130.48, 130.81, 133.60, 137.80, 139.20, 141.80, 145.01,
149.60, 150.01, 151.40, 152.99, 153.30, 160.55 (CONH). IR (KBr,
cmꢀ1): 3335, 3092, 2986, 2824, 1659, 1578, 1522, 827, 762.
113.56, 120.38, 124.12, 124.67 (q, CF3), 125.27, 125.32, 127.83, 127.96,
128.44, 130.09, 133.73, 134.47, 134.85, 137.58, 142.28, 144.52, 144.83,
146.45, 148.04, 152.43, 153.89, 164.72 (NHCO). IR (KBr, cmꢀ1): 3437,
351, 2992, 2839, 1595, 1570, 1528, 845, 766.
4.1.6.17. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-N-(4-chloro-3-
(trifluoromethyl)phenyl)-4-fluorobenzamide (20o). Yellow solid
(91 mg, 39.3%) was obtained from 13o. Mp: 319e321 ꢂC. HRMS
(ESIþ) m/z calculated for C24H14ClF4N7O [MþH]þ 528.0957; found,
4.1.6.13. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-chloro-N-(4-
chloro-3-(trifluoromethyl)phenyl)benzamide (20k). Yellow solþid
(93 g, 35.6%) was obtained from 13k. Mp: 313e315 ꢂC. HRMS (ESI )
528.0960. 1H NMR [300 MHz, DMSO-d6]:
d
7.08 (1H, dd, J ¼ 4.8 Hz,
J ¼ 7.8 Hz), 7.43 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.4 Hz), 7.63e7.66 (2H, m),
8.07e8.11 (1H, m), 8.37e8.38 (2H, m), 8.68 (1H, d, J ¼ 4.8 Hz), 8.96
(1H, s, H-purine), 9.23 (1H, s, H-purine), 9.80 (1H, d, J ¼ 7.8 Hz),
10.61 (1H, s, CONH), 12.88 (1H, s), 13.89 (1H, s, 9H-purine). 13C NMR
m/z calculated for
C
24H14Cl2F3N7O [MþH]þ 544.0662; found,
544.0666. 1H NMR [300 MHz, DMSO-d6]:
d
7.19 (1H, dd, J ¼ 4.8 Hz,
J ¼ 8.1 Hz), 7.61 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.1 Hz), 7.73 (2H, d,
J ¼ 8.4 Hz), 8.11 (1H, dd, J ¼ 2.7 Hz, J ¼ 8.4 Hz), 8.39 (1H, d,
J ¼ 2.7 Hz), 8.47 (1H, dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.74 (1H, s), 9.14 (1H,
s, H-purine), 9.38 (1H, s, H-purine), 9.87 (1H, d, J ¼ 7.2 Hz), 10.69
(1H, s, CONH), 13.19 (1H, s), 13.89 (1H, s, 9H-purine). 13C NMR
(75 MHz, DMSO-d6):
d
113.73, 114.37, 114.70 (J ¼ 7.0 Hz), 118.79 (q,
CF3), 121.21 (J ¼ 8.1 Hz), 124.11, 124.54, 124.75, 126.32, 126.73,
128.90, 129.01, 129.42, 130.66, 131.79, 138.68, 141.64, 144.94, 149.59
(J ¼ 6.7 Hz), 151.44, 153.15 (J ¼ 5.3 Hz), 153.46, 156.48, 165.42
(NHCO). IR (KBr, cmꢀ1): 3192, 3102, 2992, 2839, 1674, 1593, 1570,
1522, 858, 768.
(75 MHz, DMSO-d6):
d 114.19, 115.16, 118.88, 120.49, 120.89, 121.63,
123.80, 124.26, 124.87, 125.90 (q, CF3), 129.01, 129.32, 131.83, 133.39,
137.79, 138.58, 141.75, 144.83, 149.53, 149.94, 151.37, 153.11, 153.25,
165.47 (CONH). IR (KBr, cmꢀ1): 3192, 3102, 2992, 2839, 1674, 1593,
1570, 1522, 858, 768.
4.1.6.18. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)phenyl)-4-
chloro-3-(trifluoromethyl)benzamide (21a). Yellow solid (75 mg,
30.4%) was obtained from 16a. Mp: 306e308 ꢂC. HRMS (ESIþ) m/z
calculated for C24H15ClF3N7O [MþH]þ 510.1051; found, 510.1056. 1H
4.1.6.14. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-chloro-N-(3-
(trifluoromethyl)phenyl)benzamide (20l). Yellow solid (80 mg,
27.6%) was obtained from 13l. Mp: 300e302 ꢂC. HRMS (ESIþ) m/z
calculated for C24H15ClF3N7O [MþH]þ 510.1051; found, 510.1055. 1H
NMR [300 MHz, DMSO-d6]:
d
7.03 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
7.32 (1H, t, J ¼ 7.8 Hz), 7.46 (1H, d, J ¼ 7.8 Hz), 7.56 (1H, d, J ¼ 7.8 Hz),
7.92 (1H, d, J ¼ 8.7 Hz), 8.24 (1H, s), 8.27 (1H, dd, J ¼ 2.1 Hz, 8.7 Hz),
8.37 (1H, dd, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.40 (1H, d, J ¼ 1.8 Hz), 8.69 (1H,
s, H-purine), 9.09 (1H, s, H-purine), 9.76 (1H, d, J ¼ 7.8 Hz), 10.51
(1H, s, NHCO), 12.55 (1H, s), 13.82 (1H, s, 9H-purine). 13C NMR
NMR [300 MHz, DMSO-d6]:
d
7.19 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz),
7.47 (1H, d, J ¼ 7.8 Hz), 7.59e7.63 (2H, m), 7.73 (1H, d, J ¼ 8.1 Hz),
8.05 (1H, d, J ¼ 8.1 Hz), 8.27 (1H, s), 8.47 (1H, dd, J ¼ 1.8 Hz,
J ¼ 4.8 Hz), 8.75 (1H, s, H-purine), 9.13 (1H, s, H-purine), 9.38 (1H, s,
J ¼ 1.8 Hz), 9.86 (1H, d, J ¼ 1.8 Hz), 10.69 (1H, s, CONH), 13.18 (1H, s),
(75 MHz, DMSO-d6): d 112.42, 112.92, 113.92, 114.46, 116.22, 121.56,
123.73, 127.01 (q, CF3), 128.68, 129.42, 131.75, 133.30. 133.80, 134.18,
138.96, 140.79, 141.68, 144.63, 149.78, 150.02, 152.07, 152.98, 153.94,
163.03 (NHCO).
13.98 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 114.19,
115.02, 116.40, 119.86, 120.47, 120.88, 123.05, 123.77, 125.22, 125.76,
128.91, 129.25, 129.55 (q, CF3), 133.67, 137.81, 139.92, 141.78, 144.63,
149.43, 149.83, 151.45, 152.83, 153.29, 165.54 (CONH). IR (KBr,
cmꢀ1): 3090, 2988, 2922, 2847, 1666, 1607, 1564, 1526, 1332, 924,
793.
4.1.6.19. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-4-chloro-3-(trifluoromethyl)benzamide
(21b).
Yellow solid (71 mg, 28.0%) was obtained from 16b. Mp:
304e305 ꢂC. HRMS (ESIþ) m/z calculated for C25H17ClF3N7O
[MþH]þ 524.1208; found, 524.1211. 1H NMR [300 MHz, DMSO-d6]:
4.1.6.15. 3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-chloro-N-(3-
isopropylphenyl)benzamide (20m). Yellow solid (6þ9 mg, 29.0%) was
obtained from 13m. Mp: 170e172 ꢂC. HRMS (ESI ) m/z calculated
for C26H22ClN7O [MþH]þ 484.1647; found, 484.1655. 1H NMR
d
2.54 (3H, s, ArCH3), 7.07 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.45 (1H, d,
J ¼ 9.0 Hz), 7.59 (1H, d, J ¼ 9.0 Hz), 7.71 (1H, d, J ¼ 9.0 Hz), 8.12 (1H,
dd, J ¼ 2.4 Hz, J ¼ 9.0 Hz), 8.36e8.39 (2H, m), 8.73 (1H, s), 8.94 (1H,
s, H-purine), 9.10 (1H, s, H-purine), 9.85 (1H, s), 10.56 (1H, s, NHCO),
12.45 (1H, s), 13.86 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-
[300 MHz, DMSO-d6]:
d
1.23 (6H, d, (CH3)2CH, J ¼ 6.9 Hz), 2.88e2.93
(1H, m, (CH3)2CH), 7.00 (1H, d, J ¼ 7.5 Hz), 7.18 (1H, dd, J ¼ 4.8 Hz,
J ¼ 7.8 Hz), 7.27 (1H, t, J ¼ 7.8 Hz), 7.57e7.63 (2H, m), 7.67e7.70 (2H,
m), 8.47 (1H, dd, J ¼ 1.8 Hz, J ¼ 4.8 Hz), 8.75 (1H, s), 9.14 (1H, s, H-
purine), 9.32 (1H, s, H-purine), 9.86 (1H, d, J ¼ 7.5 Hz), 10.23 (1H, s,
CONH), 13.15 (1H, s), 13.89 (1H, s, 9H-purine). 13C NMR (75 MHz,
d6):
d
18.75 (CH3), 113.15, 113.75 (d, J ¼ 2.9 Hz), 114.93, 123.94,
124.77, 127.00 (q, CF3), 129.37, 129.89, 131.69, 132.28, 133.26, 133.65,
134.26, 136.61, 138.84, 139.11, 139.29, 141.70, 144.74, 149.83, 152.03,
153.08, 154.16, 166.18 (NHCO).
DMSO-d6):
d 23.80 (CH(CH3)2), 33.48 (CH(CH3)2), 114.13, 115.12,
117.97, 118.33, 120.47, 120.92, 121.75, 125.41, 128.41, 128.90, 129.42,
134.35, 137.64, 139.05, 141.76, 144.90, 148.78, 149.59, 149.97, 151.39,
4.1.6.20. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-(trifluoromethyl)benzamide (21c). Yellow solid

594
W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
(50 mg, 20.6%) was obtained from 16c. Mp: 293e294 ꢂC. HRMS
(ESIþ) m/z calculated for C25H18F3N7O [MþH]þ 490.1598; found,
(2H, m), 8.35 (1H, dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.64 (1H, d, J ¼ 2.1 Hz),
8.71 (1H, s, H-purine), 9.08 (1H, s, H-purine), 9.80 (1H, s), 10.02 (1H,
s, CONH), 12.27 (1H, s), 13.83 (1H, s, 9H-purine). 13C NMR (75 MHz,
490.1591. 1H NMR [300 MHz, DMSO-d6]:
d 2.44 (3H, s, ArCH3), 7.03
(1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.25 (1H, d, J ¼ 8.1 Hz), 7.47 (1H, d,
J ¼ 8.1 Hz), 7.81 (1H, t, J ¼ 7.8 Hz), 7.96 (1H, d, J ¼ 7.8 Hz), 8.27e8.36
(3H, m), 8.67 (1H, d, J ¼ 1.6 Hz), 8.71 (1H, s), 9.08 (1H, s, H-purine),
9.79 (1H, s), 10.42 (1H, s, CONH), 12.29 (1H, s), 13.83 (1H, s, 9H-
DMSO-d6): d 18.27 (CH3), 55.27 (OCH3), 112.81, 112.98, 113.69,
114.03, 115.02, 117.14, 119.85, 123.71, 129.42, 129.39, 129.82, 136.48,
137.06, 138.93, 141.68, 144.66, 149.94, 149.98, 152.13, 153.03, 154.24,
159.10, 164.98 (NHCO).
purine). 13C NMR (75 MHz, DMSO-d6):
d 18.26 (CH3), 113.08, 113.69,
113.94, 115.00, 120.73, 122.89, 123.90, 124.24 (d, J ¼ 2.3 Hz), 125.06,
127.23, 127.82, 129.10 (q, CF3), 129.86, 131.76, 135.95, 136.78, 139.07,
141.69, 144.55, 149.87 (d, J ¼ 3.7 Hz), 152.12, 152.94, 154.22, 163.76
(NHCO).
4.1.6.25. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-(dimethylamino)benzamide (21h). Yellow solid
(70 mg, 31.0%) was obtained from 16h. Mp: 242e245 ꢂC. HRMS
(ESIþ) m/z calculated for C26H24N8O [MþH]þ 465.2146; found,
465.2145. 1H NMR [300 MHz, DMSO-d6]:
d 2.42 (3H, s, ArCH3), 3.00
4.1.6.21. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-cyanobenzamide (21d). Yellow solid (76 mg,
34.1%) was obtained from 16d. Mp: 303e305 ꢂC. HRMS (ESIþ) m/z
calculated for C25H18N8O [MþH]þ 447.1676; found, 447.1680. 1H
(6H, s, (CH3)2N), 6.90e6.93 (1H, m), 7.02 (1H, dd, J ¼ 4.8 Hz,
J ¼ 7.8 Hz), 7.20e7.33 (4H, m), 7.43 (1H, dd, J ¼ 2.1 Hz, J ¼ 8.1 Hz),
8.34 (1H, dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.61 (1H, d, J ¼ 2.1 Hz), 8.71 (1H,
s, H-purine), 9.08 (1H, s, H-purine), 9.82 (1H, dd, J ¼ 2.1 Hz,
J ¼ 7.8 Hz), 10.05 (1H, s, CONH), 12.26 (1H, s), 13.85 (1H, s, 9H-pu-
NMR [300 MHz, DMSO-d6]:
d 2.50 (3H, s, ArCH3), 7.04 (1H, dd,
rine). 13C NMR (75 MHz, DMSO-d6):
d 18.26 (CH3), 40.09 (N(CH3)2),
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.25 (1H, d, J ¼ 8.1 Hz), 7.49 (1H, dd, J ¼ 2.1 Hz,
J ¼ 8.1 Hz), 7.75 (1H, t, J ¼ 7.8 Hz), 8.04e8.07 (1H, m), 8.26e8.28
(1H, m), 8.36 (1H, dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.43 (1H, d, J ¼ 1.5 Hz),
8.68 (1H, d, J ¼ 2.1 Hz), 8.72 (1H, s, H-purine), 9.09 (1H, s, H-purine),
9.82 (1H, s), 10.38 (1H, s, NHCO), 12.32 (1H, s), 13.85 (1H, s, 9H-
111.24, 112.95, 113.67, 114.18, 115.04, 115.13, 115.23, 123.63, 128.75,
129.63, 129.78, 135.82, 137.28, 138.88, 141.68, 144.67, 149.98, 149.99,
150.22, 152.14, 152.98, 154.28, 165.94 (NHCO).
purine). 13C NMR (75 MHz, DMSO-d6):
d 18.31 (CH3), 111.39, 113.07,
4.2. In vitro B-RafV600E kinase assay
113.57, 113.75, 114.68, 118.33 (CN), 123.79, 129.55, 129.64, 129.90,
131.25, 132.48, 134.71, 136.03, 136.74, 139.05, 141.70, 144.61, 149.86,
149.92, 152.06, 152.98, 154.15, 163.33 (NHCO).
Activity of full length B-RafV600E was determined using Hot-
SpotSM kinase assay which was performed by Reaction Biology
Corp. (Malvern PA). 5 nM of human GST-tagged B-RafV600E protein
4.1.6.22. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-4-chloro-3-methylbenzamide (21e). White-off solid
(84 mg, 35.7%) was obtained from 16e. Mp: 301e302 ꢂC. HRMS
(ESIþ) m/z calculated for C25H20ClN7O [MþH]þ 470.1491; found,
(AA416-766) (Invitrogen, Cat# PV3849) was mixed with 20 mM of
the substrate His6-Tagged Full-length Human MEK1 (K97R) (Re-
action Biology Corp.) in reaction buffer (20 mM Hepes pH 7.5,
10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM
Na3VO4, 2 mM DTT, 1% DMSO) at room temperature, then com-
pounds dissolved in 100% DMSO at indicated doses (starting at
470.1490. 1H NMR [300 MHz, DMSO-d6]:
d 2.21 (3H, s, ArCH3), 2.34
(3H, s, ArCH3), 7.25 (1H, d, J ¼ 8.4 Hz), 7.49 (1H, dd, J ¼ 2.1 Hz,
J ¼ 8.4 Hz), 7.56 (1H, d, J ¼ 7.8 Hz), 8.06 (1H, d, J ¼ 7.8 Hz), 8.27 (1H,
dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.36 (1H, dd, J ¼ 2.1 Hz, J ¼ 4.8 Hz), 8.43
(1H, d, J ¼ 1.8 Hz), 8.68 (1H, d, J ¼ 1.8 Hz), 8.72 (1H, s, H-purine),
9.08 (1H, s, H-purine), 9.80 (1H, s), 10.38 (1H, s, NHCO), 12.32 (1H,
30
mixture by Acoustic technology (Echo550; nanoliter range), incu-
bate for 20 min at room temperature. After 10 -ATP
33P-
(specific activity 10 Ci/ L) (P-ERKin Elmer, NEG302H001 MC) was
mM with 3-fold dilution) was delivered into the kinase reaction
mM
g
s), 13.85 (1H, s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 17.67
m
m
added to initiate the reaction, the reactions were carried out at
25 ꢂC for 120 min. The kinase activities were detected by filter-
binding method. IC50 values and curve fits were obtained by
Prism (GraphPad Software).
(CH3), 19.83 (CH3), 113.56, 114.02, 119.39, 120.48, 121.06, 122.65,
127.31, 128.74, 129.44, 129.91, 131.94, 132.85, 135.29, 138.21, 139.14,
141.72,144.64,149.90,149.94,151.98,152.98,154.13,165.80 (NHCO).
4.1.6.23. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-chlorobenzamide (21f). Yellow solid (93 mg,
39.5%) was obtained from 16f. Mp: 311e313 ꢂC. HRMS (ESIþ) m/z
calculated for C24H18ClN7O [MþH]þ 456.1334; found, 456.1331. 1H
4.3. Antiproliferative assay in A375 cell lines in vitro
EnoGeneCell™ Counting Kit-8 (Cologne, Germany) was used to
evaluate the inhibition effect on the growth of Human A375 mel-
anoma cells. After diluting to 5 ꢃ 104 cells mLꢀ1 with the complete
NMR [300 MHz, DMSO-d6]:
d 2.43 (3H, s, ArCH3), 7.04 (1H, dd,
J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.24 (1H, d, J ¼ 8.4 Hz), 7.47 (1H, dd, J ¼ 2.1 Hz,
J ¼ 9.0 Hz), 7.57 (1H, t, J ¼ 8.4 Hz), 7.66 (1H, d, J ¼ 9.0 Hz), 7.94 (1H, d,
J ¼ 7.8 Hz), 8.03 (1H, d, J ¼ 1.8 Hz), 8.36 (1H, dd, J ¼ 2.1 Hz,
J ¼ 4.8 Hz), 8.67 (1H, d, J ¼ 1.8 Hz), 8.72 (1H, s, H-purine), 9.09 (1H, s,
H-purine), 9.82 (1H, s), 10.30 (1H, s, NHCO), 12.30 (1H, s), 13.84 (1H,
medium, 100 mL of human A375 melanoma cells suspension was
added to each well of 96-well culture plates and cultured in DMEM/
10% fetal bovine serum for 24 h, with 5% CO2 water saturated at-
mosphere at 37 ꢂC. The synthesized compounds, Sorafenib and
Verumrafenib were initially prepared at 20 mM in DMSO. Aliquots
s, 9H-purine). 13C NMR (75 MHz, DMSO-d6):
d 18.79 (CH3), 113.53,
114.24, 114.29, 115.34, 124.29, 126.95, 127.88, 129.88, 130.36, 130.74,
131.64, 133.60, 137.35, 137.52, 139.48, 142.20, 145.17, 150.41, 150.46,
152.60, 153.35, 154.69, 164.44 (NHCO).
(200
and prepared into 10 doses with two-fold dilutions. Aliquots
(100 L) of each dilution were added to the wells, giving doses
ranging from 0.196 M to 100 M. A negative control group and
mL) were diluted into 20 mL culture medium giving 200 mM,
m
m
m
4.1.6.24. N-(3-((3-(9H-purin-6-yl)pyridin-2-yl)amino)-4-
methylphenyl)-3-methoxybenzamide (21g). Yellow solid (10þ0 mg,
44.4%) was obtained from 16g. Mp: 275e277 ꢂC. HRMS (ESI ) m/z
calculated for C25H21N7O2 [MþH]þ 452.1839; found, 452.1838. 1H
vehicle control group each were also set. After further incubated at
37 ꢂC for another 72 h in a humidified atmosphere with 5% CO2, the
cell viability was assessed by CCK-8 treatment and carried out
strictly according to the manufacturer instructions (EnoGeneCell).
The absorbance at 450 nm was recorded using MK3 Thermo
microplate reader. Then IC50 values were calculated using Graph-
Pad Prism 5 Software.
NMR [300 MHz, DMSO-d6]: d 2.43 (3H, s, ArCH3), 3.81 (3H, s, CH3O),
7.03 (1H, dd, J ¼ 4.8 Hz, J ¼ 7.8 Hz), 7.15 (1H, dd, J ¼ 1.6 Hz,
J ¼ 8.1 Hz), 7.22 (1H, d, J ¼ 8.7 Hz), 7.44 (2H, t, J ¼ 7.8 Hz), 7.50e7.57

W. Yang et al. / European Journal of Medicinal Chemistry 89 (2015) 581e596
595
4.4. p-ERK cellular assay in A375 cells and SK-MEL-2 cells
project of National Natural Science Foundation of China (No.
81102318/H3001) and A Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions. We
also express our gratitude to Dr. Yuren Wang, Dr. Jianghong Wu and
Dr. Jamie Planck (Reaction Biology Corporation, Pennsylvania, USA)
for their helpful support in biological evaluation.
A375 and SK-MEL-2 human melanoma cell line were both ob-
tained from American Type Culture Collection (Manassas, VA).
A375 cells were seeded at 0.15 ꢃ 106 cells/well into 24 well plates in
DMEM medium and SK-MEL-2 cells were grown in EMEM medium
supplemented with 10% heat-inactivated fetal bovine serum (FBS),
100 mg/mL penicillin, and 100 mg/mL streptomycin. The cells were
Abbreviations
treated with compounds or DMSO for 90 min. The final DMSO
concentration for each culture sample was 0.5%. After treatment,
the culture medium was removed and the cells were washed once
MAPK
MEK
ERK
mitogen-activated protein kinase
mitogen-activated protein kinase kinase
extracellular signal-regulated kinase
with ice cold PBS. 60 mL of SDS sample buffer (62.5 mM TriseHCl pH
6.8, 2% SDS, 10% Glycerol, 0.01% bromophenol red, 50 mM DTT) was
added to each well of the 24 well plates to lyse the cells. The lysed
cell samples were transferred to eppendorf tubes. The samples
were then sheared using 27 gauge needles to shear DNA in the
V600E a substitution of a glutamic acid for valine at residue 600
DFG
wt
a conserved amino acid sequence of D594 F595 and G596
wild-type
FDA
SAR
EDCI
Food and Drug Administration
structureeactivity relationships
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
whole cell lysates. 15 mL of cell lysate samples were separated by
SDS-PAGE with 10% Bis-Tris gel and transferred onto nitrocellulose
membranes by iBlot dry blotting system (Life Technologies).
The membranes were blocked with 3% non-fat milk for 2 h and
probed with primary anti-phospho-ERK1/2 antibody for overnight.
Alexa Fluor633 goat anti rabbit IgG was used to detect the primary
antibody. The membranes were scanned by Typhoon 9410 (GE
Healthcare Life Sciences, Pittsburgh, PA). The blots were then
stripped with a stripping buffer (100 mM Glycine, pH 2.5, 200 mM
NaCl, 0.1% Tween 20 and 0.1% beta-mercaptoethanol) and reprobed
with anti-ERK1/2 antibody and the Alexa Fluor633 goat anti rabbit
IgG secondary antibody to get the ERK protein expression signal for
each sample. In some cases, separate gels were run and the blots
were probed anti-ERK1/2 antibody directly. The specific signals of
bands of interest were quantified by Gel-Pro Analyzer. The IC50
values of the testing compounds were calculated using the
GraphPad Prism 5 program based on a sigmoidal doseeresponse
equation.
HOBt
DMF
THF
N-hydroxybenzotriazole
N,N-dimethylformamide
tetrahydrofuran
PdCl2(dppf) 1,10-bis-(diphenylphosphino)ferrocene-palladium(II)
dichloride
LiHMDS lithium hexamethyldisilazide
EtOAc
THP
TsOH
ESI
HRMS
SD
ethyl acetate
tetrahydropyran
p-toluenesulfonic acid
electrospray ionization
high-resolution mass spectroscopy
standard deviation
SEM
TLC
standard error of the mean
thin-layer chromatography
DMSO dimethyl sulfoxide
FBS fetal bovine serum
DMEM Dulbecco's modified eagle medium
SD solid dispersion
4.5. In vivo A375 xenograft assay protocol in Balb/c-nu nude mice
Adult female Balb/c-nu nude mice (4e6 weeks of age and
16e17 g of weight) were purchased from Shanghai Sciple-Rubicam
experimental animals Ltd. with License Number of SCXK (Shanghai)
2008e0016, housed in SPF animal facility with a 12-h light/dark
cycle, and provided with rodent chow and tap water adlibitum. All
animal care and experimental procedures were reviewed and
approved by our Institutional Animal Care and Use Committee.
Human melanoma A375 cells were purchased from ATCC. Cells
were expanded in culture at 37 ꢂC humidified atmosphere con-
taining 5% CO2 using DMEM supplemented with 10% fetal bovine
serum (Gibco). All cells were kept in culture for less than 10 pas-
sages. Cells growing at 80% confluence were harvested for im-
plantation using 0.25% trypsin and re-suspended in PBS at a
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.10.039.
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