Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2012.08.014

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino- 3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI₅₀ values ranging from 0.33 to 3.45 μM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC₅₀ values of 2.2-3.0 μM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.

Full text of DOI:10.1016/j.bmcl.2012.08.014

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6224–6228
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of diarylamines and diarylethers as cytotoxic
antitumor agents
Xiao-Feng Wang a, Xing-Tao Tian a, Emika Ohkoshi b, Bingjie Qin a, Yi-Nan Liu b, Pei-Chi Wu b,
Mann-Jen Hour b,e, Hsin-Yi Hung b, Keduo Qian b, Rong Huang b, Kenneth F. Bastow c, William P. Janzen d,
Jian Jin d, Susan L. Morris-Natschke b, Kuo-Hsiung Lee b,f, Lan Xie a,
a Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China
⇑
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
c Division of Medicinal Chemistry & Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 25799, USA
d Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 25799, USA
e School of Pharmacy, China Medical University, Taichung, Taiwan
f Chinese Medicine Research and Development Center, China Medical University & Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cyto-
toxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were
designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human
tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI50 values ranging
Received 4 June 2012
Revised 26 July 2012
Accepted 1 August 2012
Available online 9 August 2012
from 0.33 to 3.45 lM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an
active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring)
were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-
ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential bio-
logical targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and
13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types,
Keywords:
Diarylamines
Diarylethers
Cytotoxicity
Mer tyrosine kinase
with IC50 values of 2.2–3.0 lM. Therefore, these findings provide a good starting point to optimize a
new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.
Ó 2012 Elsevier Ltd. All rights reserved.
Cancers are complex diseases that involve uncontrolled multi-
plication and spread (metastasis) of abnormal cells. Over the past
decade, advances in our understanding of the molecular biology
of tumor cells have greatly promoted the discovery of small-mole-
cule antitumor drugs targeting distinct proteins in signal transduc-
tion pathways. Imatinib (1, Gleevec), the first drug that specifically
inhibited Abl kinase,1 is used in treating chronic myelogenous leu-
kemia (CML), gastrointestinal stromal tumors (GISTs) and other
cancers. Gefitinib (2, Iressa)2 and Erlotinib (3, Tarceva),3 both epi-
dermal growth factor receptor (EGFR) tyrosine kinase inhibitors,
were approved for treatment of metastatic non-small cell lung can-
cer, pancreatic cancer, and several other types of cancer. Sorafenib
(4, Nexavar)4 was used against advanced renal cancer. Recently,
two kinase inhibitor drugs nilotinib (5) and dasatinb (6) were ap-
proved to treat chronic myeloid leukemia (CML). Even though
these drugs and some clinical candidates have provided better
treatments for cancer patients, the chemotherapeutic therapy is
still largely unsatisfactory and there remains an urgent need to de-
velop innovative agents and/or novel combinations to improve
current therapeutic effectiveness.
In an analysis of the structures of the described tyrosine kinase
inhibitors 1–6, as well as other clinical candidates, we discovered
that most of them share a basic diarylamine or diarylether func-
tionality as shown in red in Figure 1. Therefore, we initially synthe-
sized the hit compound 7 [6-chloro-2-(4-cyanophenyl) amino-3-
nitropyridine] with a similar basic structure core to serve as the
design template. Compound 7 showed significant cytotoxic activity
against a panel of human tumor cell lines (HTCL), including A549
(human lung cancer), KB (nasopharyngeal carcinoma), KB-vin (vin-
cristine-resistant KB subline), and DU145 (prostate cancer) cell
lines, with low micromolar GI50 values of 2.11 to 6.12 lM. Accord-
ingly, 7 was modified by the strategy shown in Figure 2. We first
changed substituents R1 on the B-ring and R2 on the A-ring, and
subsequently conducted isosteric replacements for the A-ring and
the linker (Y). Totally, 30 diarylamines (9a–k, 10a–c, 11a–e) and
diarylethers (12, 13a–e, 14a–f) were synthesized and evaluated
against a human tumor cell line (HTCL) panel, including A549,
⇑
Corresponding author. Tel./fax: +86 10 6931690.
E-mail address: lanxieshi@yahoo.com (L. Xie).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.014

X.-F. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6224–6228
6225
Cl
F
NC
N
N
Thr315
HN
NH
N
O
NH
N
O
NH
N
N
O
N
O
O
N
N
N
O
O
O
1
2
(Gleevec, 2001)
(Iressa, 2003)
F3C
3 (Tarceva, 2004)
Cl
O
N
O
N
NHCH3
N
HN
O
N
Cl
F3C
HN
S
NH
HN
N
NH
O
N
N
N
O
N
N
H
N
N
HO
4
5
6
(Dasatinib, 2010)
(Nevavar, 2005)
(Nilotinib, 2007)
Figure 1. Anticancer drugs 1–6 targeting different kinases.
CN
SAR
R1
over longer times, and 9j was obtained in a lower 21% yield. The
synthesis of 5-nitropyridine analog 10b was accomplished by
using the polar aprotic solvent DMSO and K2CO3 at 110 °C for 4 h
(Method C).8 With DMF as the solvent, diphenylamines 11a–11e
were produced easily in high yields of 80–94% in the presence of
Et3N or t-BuOK at room temperature for 30 min (Method D).9,10
Moreover, mild conditions were also used to prepare 9h and
13a–d in moderate to high yields (65–91%) in the presence of
K2CO3 in t-BuOH at room temperature for 12–24 h (Method E).11
Scheme 2 illustrates the syntheses of compounds 14a–f with dif-
ferent substituents at the 3-position on the pyridine ring. The 3-ni-
tro group in 13b was first reduced to an amino group in 14a by
using literature methods.12 Subsequently, diazotization by NaNO2
in the presence of H2SO4, HCl, or HBr and treatment of the resulting
diazonium salt with Cu2O/EtOH, CuCl, CuBr, or KI, respectively,
afforded the corresponding 14b and halogenated compounds
14c–e, that is, the diazo group was replaced by H, Cl, Br, or I,
respectively. Subsequently, the iodo compound 14e was treated
with cuprous cyanide to produce the cyano compound 14f in a
96% yield. All target compounds were identified by NMR and MS
spectroscopic data.13
B
B
HN
Y
A
NH, O
N, CH
X
N
Y
NH
NO2
9, 10
N
X
A
11 CH NH
12 CH
13, 14
O
O
Cl
R2
N
7 (Hit)
Figure 2. Structural modification and target compounds 9–14.
KB, KBvin, DU145, and K652. Furthermore, active new leads were
screened against 72 kinases in a high throughput screening plat-
form to explore potential biologic targets. Herein, we present
chemical synthesis of the new diarylamines and diarylethers, their
cytotoxic activities, SAR results, and potential biologic target of
new leads.
Chemistry
Target compounds 9–13 were synthesized as shown in
Scheme 1. Halogenated mono- or di-nitropyridines (8a–8d) and
dinitrobenzene (8e), and various 4-substituted anilines or phenols
were commercially available. Because the nucleophilicity of a
substituted aniline or phenol affects the coupling reaction, target
compounds were synthesized under different reaction conditions
(see Scheme 1). The substitution reactions of 2,5-dichloro-3-ni-
tro-pyridine 8a with a nucleophilic aniline or phenol took place
preferentially at the chloride on the ortho-position to the nitro
group5 to give 9a–k and 13a–e. The coupling between 8a and ani-
lines substituted with an electron-donating group(s) proceeded
easily in the presence of NaHCO3 in EtOH at room temperature
(Method A, see 9a–e).6 A microwave-assisted method (Method
B)7 was also applied to the coupling reaction for synthesizing
compounds 9f–g, 9i–k, 10a, 10c, 12, and 13e in the presence of
anhydrous potassium carbonate in t-BuOH at 120–190 °C for 10–
40 min, achieving moderate to high yields. However, the coupling
reaction of an aniline with a strong electron-withdrawing carbox-
ylic group (CO2H) was more difficult, even at higher temperature
The 30 newly synthesized diarylamines (9–11 series) and diary-
lethers (12–14 series) were initially screened for cytotoxic activity
against a HTCL panel (A549, KB, KB-vin, and DU145 cancer cell
lines)14 with paclitaxel and 7 serving as reference compounds.
The in vitro anticancer activity (GI50) was determined using the
established sulforhodamine B (SRB) method.15 Target compounds
and their biological data are summarized in Tables 1 and 2. Among
them, the four most potent new compounds, 11e, 12, 13a, and 13b,
showed lower micromolar GI50 values of 0.33–3.45
lM against the
HTCL panel and were more potent than 7 (GI50 2.11–6.12
lM).
The impact of the substituent (R1) at the para-position of the
phenyl ring (B-ring) in 7 was explored by synthesizing and testing
series 9 compounds (9a–k) for cytotoxic activity. Compared with
9d, which has an unsubstituted phenyl B-ring (GI50 >17 lM), most
remaining compounds in this series showed higher potency, indi-
cating that a para-substituent on the B-ring might be necessary
for cytotoxic activity. Compound 9a and 9b with a 4-methoxy or

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X.-F. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6224–6228
NO2
O
NO2
R1
Cl
R1
Cl
H2N
HO
NO2
Cl
R1
b or e
a, b, or e
Cl
N
N
N
H
R1
N
13a-e (R1 see Table 2)
8a
9a-k (R1 see Tables 1-2)
NH2
NO2
O2N
NO2
b
c
N
N
H
OMe
N
N
H
OMe
N
Br
OMe
10b
8b 3-NO2
8c 5-NO2
10a
YH
O2N
Cl
NO2
Y
O2N
NO2
O2N
R2
NO2
Cl
b
b or d
R1
N
N
H
OMe
X
R1
Y = NH; 11a-e (R1 see Table 2)
Y = O; 12 R1 = CN
10c
8d X =N, R2 = H
8e X = CH, R2 = Cl
Scheme 1. Synthesis of target compounds 9–13. (a) NaHCO3/EtOH, rt, 12–40 h; (b) microwave irradiation 120–190 °C, K2CO3/t-BuOH, 10–40 min; (c) K2CO3, DMSO, 110 °C,
N2, 4 h; (d) Et3N for 11a–c or t-BuOK for 11d–e in DMF, rt, 30 min; (e) K2CO3/t-BuOH, rt, 12–24 h.
NO2
O
NH2
O
R
O
a
b
14b R = H
14c R = Cl
14d R = Br
14e R = I
Cl
N
Cl
N
Cl
N
c
14f R = CN
13b
14a
OMe
OMe
OMe
Scheme 2. Synthesis of target compounds 14a–e. (a) NaBH4, NiCl2ꢀ6H2O/MeOH, 0–45 °C, 52%; (b) 14b: H2SO4/NaNO2, CuO2/EtOH, 50 °C; 14c: HCl/NaNO2, CuCl/HCl, 50 °C;
14d: 48%HBr/NaNO2, CuBr/HBr, 80 °C; 14e: H2SO4/NaNO2, KI, NaHSO3, 50 °C; (c) CuCN, Pd(PPh3)4, I2/DMF, N2, 100 °C, 10 h.
compounds substituted with halogens (F, Cl, I, OCF3) or carboxylic
acid (COOH) or ester groups (COOMe) were less active than 7
Table 1
Inhibitory activity of 9a–n against HTCL panel
against all tested tumor cell lines, see 9e–h and 9j–k. Thus, para-
cyano (7) and para-alkoxy substituents on the B-ring might favor
enhanced cytotoxic activity. Compounds with para-alkyl substitu-
NO2
ents (9c, methyl; 9i, isopropyl) were generally inactive; however,
R1
B
Cl
N
N
H
interestingly, 9i exhibited significant and selective inhibition of
A549 cell growth (GI50 1.47 M).
9
l
Next, modification of the A-ring was explored (Table 2). While
9a (R2 = 6-Cl, R3 = 3-NO2) was active, 10a (R2 = H, R3 = 3-NO2),
which lacks the 6-chloro (meta-chloro) group on the A-ring, was
inactive, suggesting that this group is a fundamental cytotoxic
pharmacophore. Furthermore, both 10b (5-NO2) and 10c (3,5-
NO2) were also inactive. The next modification involved replacing
the pyridine (A-ring) by a benzene ring to produce a series of
diphenylamine compounds (11a–11e). These analogs have a com-
mon 6-chloro-3,5-dinitrophenyl moiety (A-ring) and different
substituents on the B-ring. Similarly to the series 9 compounds,
p-methyl substituted analog 11c showed the lowest potency, while
analog 11e (p-cyanophenyl) showed broad cytotoxicity against the
a
Compd
R1
GI50
(
l
M)
A549
KB
KB-vin
DU145
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
7
OMe
OEt
Me
H
F
13.5
15.8
21.6
23.8
21.2
20.4
27.2
9.98
1.47
19.6
7.19
3.26
18.1
20.8
9.57
2.40
2.26
13.9
17.7
5.88
14.7
23.2
9.05
13.0
27.4
10.5
2.11
0.948
3.83
2.94
15.9
20.1
11.9
16.2
46.6
13.1
14.7
25.3
4.16
3.53
0.00255
Cl
I
15.5
29.3
8.03
13.9
29.1
10.2
OCF3
CHMe2
COOH
COOMe
CN
9.07
6.12
0.00489
2.73
0.00265
HTCL panel (GI50 0.33–3.45 lM). In addition, the four more active
series 11 compounds showed different cell line profiles. Analogue
Paclitaxelb
a
11a (para-OMe) was especially potent against DU145 (GI50
GI50 is concentration that inhibits 50% human tumor cell growth, and the values
were averaged from at least three independent experiments.
0.93
KB-vin (GI50 1.37 and 1.34
DU145 (GI50 8.32 and 8.29
eight-fold more potent against A549 (GI50 2.57
remaining tumor cell lines, and 11e (para-CN) was particularly po-
tent against drug-resistant KB-vin cell growth (GI50 0.33 M).
l
M), 11b (para-Cl) was six-fold more potent against KB and
M, respectively) than A549 and
M), 11d (para-NO2) was three- to
M) than the
b
Positive control.
l
l
l
4-ethoxy substituent, respectively, on the B-ring showed similar
potency to 7 against KB (not 9a), KB-vin, and DU145, and both
compounds were more potent against drug-resistant KB-vin (GI50
l
Therefore, the para-group on the B-ring affected the selectivity
2.40 and 2.26
l
M) than KB (GI50 7.19 and 3.26
lM) cells. However,
against different cell lines.

X.-F. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6224–6228
6227
Table 2
Table 3
Inhibitory activity of 10–14 against HTCL panel
Inhibitory activity against TAM family of receptor tyrosine kinases
a
Compd
IC50
Mer
(l
M) in kinase assays
GI50 (l
M) in K562 cellsb
5
3 R3
A
Tyro-3
Axl
R2
X
Y
R1
11e
12
2.2 1.8
2.4 1.6
3.0 0.5
—
>30
>30
>30
—
>10
>30
>10
—
3.72 0.34
3.01 0.23
4.79 0.47
0.39 0.12
B
13b
a
1 (Gleevec)c
Compd B-ring
R1
Linker
A-ring
GI50
(l
M)
a
Y
X
R2 R3
A549
NAb
KB
KBvin DU145
IC50 is concentration that inhibits 50% kinase.
GI50 is concentration that inhibits 50% human chronic myelogenous leukemia
b
10a
10b
10c
11a
11b
11c
11d
11e
12
OMe
OMe
OMe
OMe
Cl
NH
N
H
H
H
3-
NO2
5-
NO2
3,5- NA
NO2
NA
NA
NA
(K562) cell growth by MTS method.
c
Positive control in the cellular assay. All values were averaged from at least two
NH
NH
NH
NH
NH
NH
NH
O
N
N
NA
NA
NA
NA
or more independent experiments.
NA
NA
NA
CH Cl 3,5- 10.7
3.44
1.37
5.92
19.7
2.42
1.83
1.05
1.50
8.31
NA
5.16
1.34
6.24
16.6
0.33
1.53
1.81
1.18
10.5
11.6
8.55
0.93
8.29
8.84
8.47
1.07
0.84
0.94
2.03
8.35
11.4
7.97
11e, 12, and 13b exhibited inhibitory activity for Mer tyrosine ki-
nase (Mer TK) with IC50 values ranging from 2.2–3.0 lM, but did
NO2
CH Cl 3,5- 8.32
NO2
CH Cl 3,5- 31.1
NO2
CH Cl 3,5- 2.57
NO2
CH Cl 3,5- 3.45
NO2
not inhibit Tyro3 and Axl (IC50 >30), two other members of the
TAM kinase family (Table 3). Because Mer TK was recently identi-
fied as a specific therapeutic target for acute lymphoblastic leuke-
mia (ALL),17 compounds 11e, 12, and 13b were further tested
against the K562 (human chronic myelogenous leukemia cell line)
using the[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazoliuinm, inner salt] (MTS) colori-
metric method18 in parallel with 1 (gleevec) as the positive
control. As expected, the three new compounds did inhibit K562
Me
NO2
CN
CN
CH Cl 3,5- 3.07
NO2
13a
13b
13c
13d
13e
CN
O
N
N
N
N
N
Cl 3-
NO2
Cl 3-
NO2
Cl 3-
NO2
Cl 3-
NO2
Cl 3-
NO2
Cl 3-H NA
Cl 3-Cl NA
Cl 3-Br NA
Cl 3-I
Cl 3-
CN
1.02
2.00
10.0
NA
OMe
OEt
O
cell growth with GI50 values ranging from 3.01–4.79
although they were less potent than 1 (0.39 M) in the same assay.
lM (Table 3),
l
O
These results indicated that Mer kinase might be a major target,
but not the only target, for these leads, because they were more po-
tent in the cellular assay than the kinase inhibitory assay. How-
ever, the current results have provided us with a new starting
point to develop selective Mer kinase inhibitors by lead optimiza-
tion. A Mer TK-selective inhibitor would be a promising option for
treatment of pediatric acute lymphocytic leukemia (ALL), other
leukemias, and possibly adult solid tumors, because Mer TK is
ectopically over-expressed at high levels in pediatric T- and B-cell
ALL, but not normal, lymphocytes,19,20 and also is associated with
the pathophysiology of tumorigenesis and thrombosis.
In conclusion, the modifications on hit 7 led to the discovery of
new diarylamine and diarylether leads, 11e, 12 and 13b, which
showed significant inhibitory activity against a human tumor cell
line panel (A549, KB, KBvin, Du145, and K652) with low micromo-
lar GI50 values ranging from 0.33–4.79
tyrosine kinase with IC50 values of 2.2–3.0
CHMe2
COOMe
O
O
9.17
8.29
14b
14c
14d
14e
14f
OMe
OMe
OMe
OMe
OMe
O
O
O
O
O
N
N
N
N
N
NA
22.7
NA
NA
12.4
NA
17.4
NA
NA
12.7
NA
NA
NA
NA
NA
NA
NA
7
CN
NH
N
Cl 3-
6.12
2.73
2.11
3.53
NO2
Paclitaxelc
0.00489 0.00265 0.948 0.00255
a
GI50 is concentration that inhibits 50% human tumor cell growth, and the values
were averaged from at least three independent experiments.
b
NA: no activity.
Positive control.
c
l
M, and also inhibited Mer
M. The current SAR re-
l
sults suggested that a meta-chloro- and ortho-nitro group on the A-
ring (pyridine or benzene) and a para-cyano or methoxy group on
the B-ring (see 7, 11e, 12 and 13a, 9a, 11a, respectively) are essen-
tial pharmacophores for cytotoxic acitivity. Meanwhile, the para-
substituent on the B-ring and the linker between aryl rings are
changeable and appear to modulate the inhibitory selectivity.
These promising results provide a good starting point to further
optimize the new leads based on the diarylamine or diarylether
structural core for developing a novel class of anticancer drugs tar-
geting Mer kinase.
Next, the NH linker was replaced by an isosteric oxygen atom
(O) to obtain diarylethers 12 and 13a–e. Compounds 12, 13a, and
13b with a para-cyano or -methoxy group on the B-ring exhibited
improved potency with GI50 values of 0.84–3.07 lM, which were
similar to those for 11e. All three diarylether compounds were
more potent than 7 against the HTCL panel. Similarly to the series
9 diarylamines, the diarylether compounds 13d and 13e with para-
isopropyl and para-carboxylate substituent in the B-ring were less
potent than 13a and 13b with para-cyano and para-methoxy
groups. However, 13c with a para-ethoxy group was also signifi-
cantly less potent than 13b, unlike the related 9b and 9a. Finally,
the 3-nitro (ortho-nitro) group of active compound 13b was sys-
temically replaced with H, Cl, Br, I, or CN to give corresponding
compounds 14b–14f, resulting in great decreased or abolished
antitumor activity against all cell lines (Table 2). Thus, the ortho-ni-
tro group on the A-ring might be necessary for optimal activity or
interaction with the biologic target.
Acknowledgments
This investigation was supported by Grants 81120108022 and
30930106 from the Natural Science Fundation of China (NSFC),
and 2006DFA33560 from the Ministry of Science and Technology
Commission in China awarded to Lan Xie and NIH Grant
CA17625-32 from the National Cancer Institute awarded to K.H.
Lee. This study was also supported in part by the Taiwan Depart-
ment of Health, China Medical University Hospital Cancer Research
Center of Excellence (DOH100-TD-C-111-005).
Subsequently, active compounds 11e, 12, 13a, and 13b were
evaluated against 72 kinases in a high throughput screening plat-
form16 to explore potential drug target(s). Notably, compounds

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X.-F. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6224–6228
After removal of solvent, the crude product was purified by column
Supplementary data
chromatography, PTLC, or recrystallization to give the corresponding pure
products.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.014. These data include MOL files and InChiKeys of the most
important compounds described in this article.
12. Schmid, S.; Röttgen, M.; Thewalt, U.; Austel, V. Org. Biomol. Chem. 2005, 3, 3408.
13. The most active new compounds: 11e: Yellow solid, mp 140–141 °C. 1H NMR d
ppm 7.41 (1H, s, ArH-6), 7.57 (2H, d, J = 8.8 Hz, ArH-20,60), 7.91 (2H, d,
J = 8.8 Hz, ArH-30,50), 8.90 (1H, s, ArH-3), 10.07 (1H, s, NH); MS m/z (%) 319
(M+1+, 100), 321 (M+3+, 23); HPLC purity 100.0%. 12: Pale yellow solid, mp
118–120 °C. 1H NMR (DMSO-d6) d 5.98 (1H, s, ArH-6), 7.12 (2H, d, J = 8.8 Hz,
ArH-20,60), 7.80 (2H, d, J = 8.8 Hz, ArH-30,50), 8.73 (1H, s, ArH-3); MS m/z (%) 318
(Mꢃ1, 100), 310 (M+1, 27); HPLC purity 99.51%. 13a: Pale yellow solid, mp
References and notes
128–130 °C. 1H NMR
d ppm 7.25 (1H, d, J = 8.4 Hz, PyH-5), 7.34 (2H, d,
1. Deininger, M.; Druker, B. J. Pharmacol. Rev. 2003, 55, 401.
2. Sordella, R.; Bell, D. W.; Haber, D. A.; Settleman, J. Science 2004, 305, 1163.
3. Eric, R.; Sandrine, F.; Jean Pierre, A. Drugs 2000, 60, 15.
4. Wilhelm, S. M.; Adnane, L.; Newell, P.; Villanueva, A.; Llovet, J. M.; Lynch, M.
Mol. Cancer Ther. 2008, 7, 3129.
5. Qin, B.; Zhou, T.; Lu, H.; Jiang, S.; Xie, L. Acta Pharm. Sin. 2009, 44, 1233.
6. Synthetic Method A: General procedure for the preparation of 9a–9e. A mixture
of 8a (1.0 mmol), aniline, or substituted aniline (1.0 mmol), and sodium
bicarbonate (1.0 mmol) in absolute EtOH (10 mL) was stirred at room
temperature for 12–40 h monitored by TLC until the reaction finished. The
mixture was poured into ice-water, pH was adjusted to 3.0 with aq HCl (2 N),
and solid crude product was collected. The corresponding products were then
purified by column chromatograph, PTLC, or re-crystallization.
J = 8.8 Hz, ArH-20,60), 7.77 (2H, d, J = 8.8 Hz, ArH-30,50), 8.41 (1H, d, J = 8.4 Hz,
PyH-4); MS m/z (%) 276 (M+H+, 100), 278 (M+3+, 33); HPLC purity 99.14%. 13b:
Pale yellow solid, mp 110 °C. 1H NMR d ppm 3.83 (3H, s, OCH3), 6.93 (2H, d,
J = 9.2 Hz, ArH-30,50), 7.10 (2H, d, J = 9.2 Hz, ArH-20,60), 7.11 (1H, d, J = 8.4 Hz,
PyH-5), 8.33 (1H, d, J = 8.4 Hz, PyH-4); MS m/z (%) 281 (M+H+, 100), 283 (M+3+,
32); HPLC purity 100.0%.
14. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Woiff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
15. Rubinstein, L. V.; Shoemaker, R. H.; Paull, R. M.; Tosini, S.; Skehan, P.; Scudiero,
D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
16. Liu, J.; Yang, C.; Simpson, C.; DeRyckere, D.; Deusen, A. V.; Miley, M. J.; Kireev,
D.; Norris-Drouin, J.; Sather, S.; Hunter, D.; Korboukh, V. K.; Patel, H. S.; Janzen,
W. P.; Machius, M.; Johnson, G. L.; Earp, H. S.; Graham, D. K.; Frye, S. V.; Wang,
X. ACS Med. Chem. Lett. 2012, 3, 129.
17. Linger, R. M. A.; DeRyckere, D.; Brandao, L.; Sawczyn, K. K.; Jacobsen, K. M.;
Liang, X.; Keating, A. K.; Graham, D. K. Blood 2009, 114, 2678.
7. Synthetic method B (microwave irradiation): General procedure for the synthesis
of 9f–9g, 9i–9k, 10a, 10c, 12, and 13e. A mixture of 8a, 8b, 8d, or 8e (1.0 mmol),
a
substituted aniline (1.0–1.5 mmol), anhydrous potassium carbonate
(2.0 mmol) in 4 mL of t-BuOH (except for 12 in DMSO), was heated at 120–
180 °C with microwave-assistance for 15–40 min with stirring. After the
reaction was finished, the mixture was poured into ice-water, pH was adjusted
to ꢁ3.0 with aq HCl (2 N) or to ꢁ10.0 with aq NaOH (5%), and solid crude
product was filtered and then purified by column chromatograph, PTLC, or
recrystallization to give pure product.
18. MTS assay: K562 cells (ATCC no. CCL243) are human myelogenous leukaemia,
obtained from Lineberger Cancer Center (UNC-CH). Cells were cultured in RPMI
1640 medium containing 25 mM HEPES and 2 mM
supplemented with 10% fetal bovine serum (Hyclone), 100 IU penicillin,
100 g/mL streptomycin, and 0.25 g/mL amphotericin B (Mediatech). Cells
L-glutamine(Mediatech),
l
l
8. Hirauchi, K.; Amano, T. Chem. Pharm. Bull. 1979, 27, 1120.
9. Bankston, D.; Dumas, J.; Natero, R.; Riedl, B.; Monahan, M. K.; Sibley, R. Org.
Process Res. Dev. 2002, 6, 777.
were maintained at 37 °C in a humidified 5% CO2 atmosphere. K562 cell
viability was determined using a colorimetric assay, Cell Titer 96R AQueous
Non-radioactive Cell Proliferation Assay (Promega, Medison, WI) according to
the manufacturer’s protocol. In brief, the cells (5 ꢂ 103 cells/well) were seeded
in 96-well plates filled with culture medium containing various concentrations
10. Synthetic Method D: Preparations of 11a–c, Et3N (2 mmol) was slowly added
into the solution of 8e (1.0 mmol) and 4-substituted aniline (1.1 mmol) in DMF
(3 mL) at below 0 °C and then was stirred at room temperature for 30 min
monitored by TLC. However, the syntheses of 11d–e were performed in the
presence of t-BuOK (2.0 mmol), rather than Et3N. After the reaction was
finished, the mixture was poured into ice-water, pH was adjusted to 6, and
solid was precipitated. The collected crude product was then recrystallized
from EtOH to afford corresponding pure products.
of samples. After 72 h of incubation, 20 lL/well of combined MTS/PMS solution
was added and incubated for 2–3 h. The absorbance was measured at 490 nm
subtracting the background at 630 nm using a Microplate Reader ELx800 (Bio-
Tek Instruments, Winooski, VT) with a Gen5 software. The number of viable
cells was counted in
a hemocytometer chamber using trypan blue dye
exclusion (Sigma). All data represent at least three independent experiments
performed in duplicate.
11. Synthetic Method E: General procedure for the synthesis of the compounds 9h,
13a–13d.
A mixture of 8a (1.0 mmol), substituted aniline/phenol (1.0–
19. Graham, D. K.; Salzberg, D. B.; Kurtzberg, J.; Sather, S.; Matsushima, G. K.;
Keating, A. K.; Liang, X.; Lovell, M. A.; Williams, S. A.; Dawson, T. L.; Schell, M. J.;
Anwar, A. A.; Snodgrass, H. R.; Earp, H. S. Clin. Cancer Res. 2006, 12, 2662.
20. Guttridge, K. L.; Luft, J. C.; Dawson, T. L. J. Biol. Chem. 2002, 277, 24057.
1.5 mmol), and anhydrous potassium carbonate (2 mmol) in t-BuOH was
stirred at room temperature for 12–24 h monitored by TLC until the reaction
completed. When the reaction finished, the mixture was poured into ice-water,
pH was adjusted to about 3.0 or 10.0 with aq HCl (2 N) or NaOH (5%),
respectively, and the mixture was then extracted with CH2Cl2 (20 mL ꢂ 3).