Discovery of 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors. Part I: Optimization of in vitro potencies and pharmacokinetic properties

Article abstract of DOI:10.1021/jm401990s

A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4′ 4-methoxyphenoxy and 4-(o-fluoropyridyl) carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC₅₀: 16.3-42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [ ³H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.

Full text of DOI:10.1021/jm401990s

Article
pubs.acs.org/jmc
Discovery of 4‑Aryl‑N‑arylcarbonyl-2-aminothiazoles as Hec1/Nek2
Inhibitors. Part I: Optimization of in Vitro Potencies and
Pharmacokinetic Properties
Ying-Shuan E. Lee,† Shih-Hsien Chuang,† Lynn Y. L. Huang,‡ Chun-Liang Lai,† Yu-Hsiang Lin,†
Ju-Ying Yang,† Chia-Wei Liu,† Sheng-chuan Yang,† Her-Sheng Lin,† Chia-chi Chang,‡ Jun-Yu Lai,§
Pei-Shiou Jian,§ King Lam,† Jia-Ming Chang,† Johnson Y. N. Lau,‡ and Jiann-Jyh Huang*,†,§
†Development Center for Biotechnology, No. 101, Lane 169, Kangning Street, Xizhi District, New Taipei City 22180, Taiwan
‡Taivex Therapeutics Corporation, 17th Floor, No. 3, Yuanqu Street, Nangang District, Taipei City 115, Taiwan
§Department of Applied Chemistry, National Chiayi University, No. 300, Syuefu Road, Chiayi City 60004, Taiwan
S
* Supporting Information
ABSTRACT: A series of 4-aryl-N-arylcarbonyl-2-aminothia-
zoles of scaffold 4 was designed and synthesized as Hec1/
Nek2 inhibitors. Structural optimization of 4 led to compound
32 bearing C-4′ 4-methoxyphenoxy and 4-(o-fluoropyridyl)-
carbonyl groups that showed low nanomolar in vitro
antiproliferative activity (IC50: 16.3−42.7 nM), high intra-
venous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and
significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell
responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1
coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity
toward cancer cells over normal phenotype cells and was inactive in a [3H]astemizole competitive binding assay for hERG
liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2
interaction.
INTRODUCTION
■
Highly expressed in cancer 1 (Hec1) is a key component of the
kinetochore that regulates the spindle check point and plays an
essential role in mitosis.1 With Nuf2, Spc24, and Spc25, it
forms a dumbbell-like heterotetramer called the Ndc80
complex, in which Hec1 and Nuf2 dimerize as a subcomplex,
with their globular domains heading toward the microtubule-
binding interface.2−4 Hec1 has microtubule-binding activity at
its N-terminal region (aa 1−196) and is responsible for proper
kinetochore−microtubule attachment.2,5,6
Hec1 possesses oncogenic properties: its hyperactivation in
transgenic mice leads to tumor formation.7 Overexpression of
Hec1 is found in various cancers8 and correlates with poor
Figure 1. Structures of 4-aryl-N-arylcarbonyl-2-aminothiazoles 1−3
and scaffold 4.
prognosis for cancer patients.9,10 RNA interference of Hec1
enhances the sensitivity of human ovarian cancer cells to
paclitaxel11 and reduces the size of induced adenocarcinomas in
nude mice.12 Because phosphorylation of Hec1 by Nek2 kinase
is essential for its mitotic function,13−15 disruption of Hec1/
Nek2 protein−protein interaction by small molecules shows
anticancer activities16,17 and may have potential for cancer
treatment.
4-Aryl-N-phenylcarbonyl-2-aminothiazole 1 (INH1, Figure
1) is the prototype Hec1 inhibitor discovered from a chemical
genetic screening.17 It specifically disrupts Hec1/Nek2
interaction via direct Hec1 binding and shows in vitro
antiproliferative activity (IC50: 8.6−11.7 μM) as well as in
vivo antitumor activity in MDA-MB-468 xenografts. Subse-
quent lead optimizations of the N-phenylcarbonyl moiety and
C-6′ position of 1 afforded compounds 2 and 3 that
demonstrated improved in vitro potency (IC50: 1.1−3.4
μM).16 Compounds 1−3 trigger the cellular responses resulting
from Hec1 inhibition, including the reduction of Nek2 protein
Received: December 28, 2013
Published: April 28, 2014
© 2014 American Chemical Society
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level and mitotic abnormalities.16,17 Although they are the only
reported Hec1/Nek2 inhibitors to date, none of them, 1−3,
have been moved into clinical trials.
For the synthesis of 4-aryl-N-arylcarbonyl-2-aminothiazoles
of scaffold 4 bearing various C-4′ alkoxy substituents (R = alkyl
in 4, Figure 1), acetophenones 10a−f were brominated by
TBABr3 in CH3CN and then reacted with thiourea in EtOH to
provide corresponding 4-aryl-2-aminothiazoles 11a−f in 57−
82% yields (Scheme 2). Acylation of 11a−f with isonicotinoyl
chloride, 2-fluoroisonicotinoyl chloride, or 2-chloroisonicoti-
noyl chloride in CH2Cl2 using DMAP as the base afforded
desired 4-aryl-N-arylcarbonyl-2-aminothiazoles 12−20 possess-
ing various C-4′ alkoxy groups in 49−99% yields.
When the structure−activity relationship of 1−3, shown in
Figure 1, was examined, compound 2, bearing C-2′ and C-6′
dimethyls, was found to be ∼5-fold more potent than
compound 1, bearing a sole C-2′ methyl (IC50: 1.7−2.5 μM
for 2 and 8.6−11.7 μM for 1). Compound 3, with a 4-
pyridylcarbonyl group, also showed ∼5-fold improved potency
(IC50: 1.1−3.4 μM) compared to 1, which has a phenylcarbonyl
group at the same position. As for the C-4′ position, no
substituent other than a methyl group has been reported on 1−
3 and their analogues in the literature.16 This information
suggested that the scaffold of 1−3 possessing C-2′ and C-6′
dimethyl and 4-pyridylcarbonyl groups could show improved
potency and that further structural optimization could be
performed on the less explored C-4′ position.
The synthesis of scaffold 4 bearing various C-4′ aryloxy
substitutents (R = aryl in 4, Figure 1) was carried out by the use
of 1-(4-amino-2,6-dimethylphenyl)ethanone 21 as the starting
material (Scheme 3). The amino group in 21 was transformed
into a chloro group by Sandermeyer-type reaction using t-
19
BuONO and CuCl2 in CH3CN to provide 1-(4-chloro-2,6-
dimethylphenyl)ethanone 22 in 76% yield. O-Arylation of
various aryl alcohols by 22 using the Pd(OAc)2/t-BuXPhos
catalytic system20 afforded 4-aryloxy-2,6-dimethylacetophe-
nones 23a−i in 19−86% yields. After reacting with TBABr3
and thiourea, 23a−i were converted to corresponding 4-aryl-2-
aminothiazoles 24a−i in 53−96% yields. Acylation of 24a−i
with isonicotinoyl chloride or 2-fluoroisonicotinoyl choride
afforded desired 4-aryl-N-arylcarbonyl-2-aminothiazoles 25−34
bearing various C-4′ aryloxy groups in 17−95% yields.
Herein, we report the design and synthesis of 4-aryl-N-
arylcarbonyl-2-aminothiazoles of scaffold 4 as Hec1 inhibitors
(Figure 1). Scaffold 4 possesses 2′,6′-dimethyl and 4-
pyridylcarbonyl groups as the core, various C-4′ alkoxy or
aryloxyl groups, and o-halo at the pyridyl group for
optimization. After evaluation, we obtained compound 32
with low nanomolar in vitro IC50 values, good pharmacokinetic
properties, and significant in vivo antitumor activity. Com-
pound 32 induced cellular responses resulting from Hec1
inhibition and was less active on normal cells, kinases, and
hERG. In our attempt to discover a preclinical candidate that
disrupts Hec1/Nek2 interaction, compound 32 serves as a
good lead for future development.
RESULTS AND DISCUSSION
■
In Vitro Potency of Scaffold 4. The in vitro potency of
the 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 (Figure
1) was evaluated against the proliferation of HeLa (cervical
cancer), K562 (leukemia), MDA-MB-468 (breast cancer), and
MDA-MB-231 (breast cancer) cancer cells. Reference com-
pound 2 was tested first. As presented in Table 1, compound 2
showed similar potency (IC50: 2.2−3.6 μM) to that reported in
the literature (IC50: 1.7−2.5 μM).16 Compound 9a, bearing
2′,6′-dimethyl and 4-pyridylcarbonyl groups, was 5.4−10.7-fold
more potent than 2. The results verified the design of scaffold 4
to improve in vitro potency. We then substituted the 4-
pyridylcarbonyl in 9a with various acyl groups to establish the
structure−activity relationship. Compound 9b with a methyl-
ene elongation to 9a showed similar potency (IC50: 0.30−0.62
μM) to that of 9a. Bioisosteric replacement of the 4-pyridyl in
9a with a 4-cyanophenyl group generated 9c with ∼2-fold
reduced potency on K562, MDA-MB-468, and MDA-MB-231
cells. Conversion of the pyridyl group in 9a to pyridyl N-oxide
afforded compound 9d with >4-fold reduced potency (IC50: 2.7
to >10 μM). Introduction of an o-chloro group to the 4-pyridyl
group in 9a provided compound 9e with slightly reduced
potency (IC50: 0.42−0.77 μM).
CHEMISTRY
■
Scheme 1 presents the synthesis of 4-mesityl-N-acyl-2-amino-
thiazoles 2 and 9a−e to verify the design of scaffold 4.
Scheme 1. Synthesis of 4-Mesityl-N-acyl-2-aminothiazoles 2
a
and 9a−e
a
Reagents and conditions: (i) BiCl3, acetyl chloride, 70 °C, 83%; (ii)
Considering the structural complexities and possible
physicochemical properties of 9a, 9b, and 9e, which possessed
similar in vitro potencies, we selected the 4-pyridylcarbonyl
group for the following optimization. Turning to the C-4′
position of scaffold 4 (Figure 1), we found that the
introduction of an alkoxy group showed enhanced antiprolifer-
ative activity (Table 2). Compounds 12, 16, 17, 19, and 20,
with the corresponding C-4′ methoxy, ethoxy, i-propoxy, i-
butoxy, and c-pentoxy groups, were more potent than 9a, which
possesses a C-4′ methyl group, and displayed IC50 values
ranging from 42.1−392 nM (cf. Table 1, IC50: 300−760 nM for
9a). The increasing size of the C-4′ substituents from the
methoxy to the c-pentoxy groups did not reduce the potency,
CuBr2, EtOAc, reflux, 95%; (iii) thiourea, EtOH, reflux, 90%; (iv)
RCOCl, DMAP, CH2Cl2, 65−87%.
Mesitylene (5) was acylated with acetyl chloride using BiCl3 as
the catalyst to provide acetyl mesitylene 6 in 83% yield.18
Compound 6 was monobrominated at the α carbon of the
acetyl group by CuBr2 in EtOAc to afford compound 7 in 95%
yield. After reacting with thiourea, 7 was converted to 4-
mesityl-2-aminothiazole 8 in 90% yield. Acylation of 8 with
various acyl chlorides gave corresponding compounds 2 and
9a−e in 65−87% yields.
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Scheme 2. Synthesis of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 12−20 Bearing C-4′ Alkoxy, C-2′ Methyl or Fluoro, and
a
o-Hydro, Fluoro, or Chloro at the Pyridyl Group
a
Reagents and conditions: (i) (a) TBABr3, CH3CN, rt, (b) thiourea, EtOH, reflux; 57−82% (two steps); (ii) isonicotinoyl chloride, 2-
fluoroisonicotinoyl chloride, or 2-chloroisonicotinoyl chloride, DMAP, CH2Cl2, 49−99%.
Scheme 3. Synthesis of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 25−34 Bearing C-4′ Aryloxy and/or Fluoro at the Pyridyl
a
Group
a
Reagents and conditions: (i) t-BuONO, CuCl2, CH3CN, 76%; (ii) aryl alcohol, Pd(OAc)2, t-BuXPhos, toluene, 100 °C, 19−86%; (iii) (a) TBABr3,
CH3CN, rt, (b) thiourea, EtOH, reflux; 53−96% (two steps); (iv) isonicotinoyl chloride or 2-fluoroisonicotinoyl chloride, DMAP, CH2Cl2, 17−95%.
indicating that the binding pocket of the Hec1 protein left a
space headed by the C-4′ substituents of scaffold 4 (Figure 1).
Accordingly, we replaced the alkoxy groups in scaffold 4 with
aryloxy groups (R = aryl in 4, Figure 1). C-4′ phenoxy
replacement of the c-pentoxy group in compound 20 generated
compound 25 with a similar potency (IC50: 94.4−236 nM) to
that of 20 (IC50: 96.0−215 nM). The introduction of a methyl
or ethyl group to the C-4 position of the phenoxy group in 25
provided compounds 26 and 27 with similar potency (IC50:
134−237 nM). However, the presence of 3,5-dimethyls at the
phenoxy group deactivated compound 28 (IC50: 727 to >1000
nM). Compound 29, with a C-4′ 4-fluorophenoxy group,
displayed similar potency (IC50: 239−280 nM) to that of 25,
26, and 27. Compound 30, with a C-4′ 4-(trifluoromethyl)-
phenoxy group, showed a ∼2-fold improvement in antiprolifer-
ative activity (IC50: 58.2−132 nM) compared to 29.
comparison with reference compound 2, 31 showed ∼100-fold
improvement (e.g., 32.0 vs 3200 nM for MDA-MB-231).
Movement of the C-4 methoxy group in 31 to the C-3 position
reduced the antiproliferative activity of 33 (IC50: 102−253
nM). Compound 34, with a 3,4-methylenedioxy moiety on the
C-4′ phenoxy group, was not as potent as 31 (IC50: 45.7−128
nM).
For further improvement of the antiproliferative activity of
scaffold 4, compounds 13−15, 18, and 32, bearing 2′,6′-
difluoro and/or o-halo atoms at the 4-pyridyl group, were
evaluated (Table 3). C-4′ methoxy-substituted compound 13,
bearing a 2′,6′-difluoro group, showed reduced in vitro activity
(IC50: 170−450 nM) in comparison with its 2′,6′-dimethyl
analogue, 12 (IC50: 99.5−172 nM, Table 2). Introduction of an
o-fluoro or o-chloro atom to the pyridyl group of 12 generated
compounds 14 and 15 that possessed slightly reduced
potencies, with IC50 values of 75.4−220 and 160−280 nM,
respectively. Compound 18, the o-fluoropyridyl analogue of 17,
showed similar potency (IC50: 42.7−167 nM) to that of 17
(IC50: 52.0−162 nM, Table 2). Introduction of a fluoro atom to
The most potent analogues of scaffold 4 in Table 2 was
compound 31, which possessed a C-4′ 4-methoxyphenoxy
functionality. Regarding the four cancer cell lines, 31 displayed
low nanomolar antiproliferative activity (32.0−48.6 nM). In
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Table 1. In Vitro Antiproliferative Activity of N-Acyl-4-mesityl-2-aminothiazoles 2 and 9a−e toward HeLa, K562, MDA-MB-
468, and MDA-MB-231 Cancer Cells
a
The IC50 values were averaged from two independent dose−response curves; variation was generally <15%.
the ortho position of the pyridyl group in 31 generated
compound 32 with comparable in vitro antiproliferative activity
(IC50: 16.3−42.7 nM). In comparison with lead compound 1,
compound 32 possessed 55−196-fold enhanced potency.
Pharmacokinetics and In Vivo Xenograft. To determine
the pharmacokinetic properties of the analogues of scaffold 4,
representative compounds 9a, 12, 17, 31, and 32 with different
substituents at the C-4′ position and/or the o-fluoro atom to
the pyridyl nitrogen were subjected to pharmacokinetic study in
Sprague−Dawley rats. The results presented in Table 4 show
that prototype 9a, bearing a C-4′ methyl group, possessed an
intravenous (IV) AUC value of 5.78 μM·h with good oral
bioavailability (41.9%). However, its clearance (CL) was high
(23.2 mL/min/kg). Compared to 9a, compound 12, possessing
a C-4′ methoxy group, showed a >5-fold improvement in the
AUC (32.7 μM·h) with reduced clearance (3.02 mL/min/kg).
Nevertheless, its Vd value was low (0.250 L/kg), and its
bioavailability was reduced (20.6%). Compound 17, with a C-4′
i-propoxy group, possessed a slightly increased AUC (44.3 μM·
h) and Vd (0.769 μM·h) as well as similar clearance and
bioavailability compared to that of 12. Compound 31,
possessing a C-4′ 4-methoxyphenoxyl group, showed a slightly
reduced AUC with increased Vd compared to that of 17.
However, its bioavailability was reduced to 12.4%.
7.4) containing 1% DMSO at 37 °C were 3.33 0.14 and 0.19
0.011 μM, respectively. As a result of this observation, we
changed the original 1% methylcellulose formulation to a 5%
DMSO, 10% cremophor, and 85% water solution to improve
the solubility of 32. The bioavailability significantly increased to
22.7% (Table 4). The result suggested that solubility might
govern the bioavailability of 32.
To realize an improved PK profile of 32, we studied the
metabolic stability of 9a and 32 in rat liver microsomes. The
half-life (t1/2) of 9a and 32 was found to be 11.0 and 340 min,
respectively. As a result, the higher metabolic stability of 32
contributed to its higher AUC (64.9 μM·h) and lower clearance
(1.22 mL/min/kg) in comparison with that of 9a (AUC: 5.78
μM·h and CL: 23.2 mL/min/kg).
Because compound 32 possessed low nanomolar IC50 values
(16.3−42.7 nM) with the highest IV and acceptable oral (PO)
AUCs, it was selected for in vivo study using nude mice bearing
MDA-MB-231 human breast cancer xenografts. As shown in
Figure 2, mice treated with 20 mg/kg of 32 for 28 days via daily
IV injection showed significant in vivo antitumor activity, with a
T/C value of 32% on day 28. Treatment of mice with 150 mg/
kg of 32 via oral gavage twice a day also showed antitumor
activity, with a T/C value of 57% on day 28, despite being less
active as an IV injection. The results reflected the different
AUCs between IV and PO routes. Meanwhile, 32 was well-
tolerated during the experiments and did not cause significant
body weight loss (<5.0%, data not shown) in the test animals.
These findings suggested that compound 32 could be a
promising anticancer lead because of its good potency and
tolerability.
Compound 32, the fluoro analogue of 31, possessed the
highest IV AUC (64.9 μM·h) among the compounds in Table
4, with a reasonable Vd (0.948 L/kg) and clearance (1.22 mL/
min/kg). However, it showed the lowest bioavailability (F =
4.6%). This result was not surprising because the o-fluoro atom
reduced the basicity of the pyridyl nitrogen, thus decreasing the
solubility of 32 in acidic gastric fluids, which then retarded its
absorption. The solubilities of 9a and 32 in PBS buffer (pH
Mechanism of Action. To confirm the mechanism of
action of promising lead 32, we performed a coimmunopre-
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Table 2. In Vitro Antiproliferative Activity of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazoles 12, 16, 17, 19, 20, 25−31, 33, and
34 Bearing a C-4′ Alkoxy or Aryloxy Group
a
The IC50 values were averaged from two independent dose−response curves; variation was generally <15%.
cipitation (Co-IP) assay using DMSO- and 32-treated K562
cells for the immunoprecipitation of Nek2 (Figure 3A). A
reduced level of coimmunoprecipitated Hec1 was observed in
cells treated with 100 nM 32 (lane 3) relative to cells treated
with DMSO (lane 2). In comparison with the literature data of
1, in which it shows activity in the Co-IP assay at a
concentration of 25 μM, the results indicated that the
disruption of Hec1/Nek2 protein−protein interaction by 32
took place in the cells with improved potency. To provide more
evidence, we analyzed the effect of 32 on the Nek2 protein level
in K562 cells by western blotting. Compound 32 led to the
degradation of Nek2 in a dose-dependent manner at
concentrations of 100 and 1000 nM; however, it had no effect
at 10 nM (Figure 3B). The same phenomenon was also
observed in MDA-MB-468 cells treated with 32 at 100 nM
(Figure 3C, lane 2). Compounds 1 and 2 are reported to
induce Nek2 degradation at concentrations of 25 and 6.25 μM,
respectively.16,17
As shown in Figure 3C, Nek2 degradation in 32-treated
MDA-MB-468 cells was blocked by the addition of proteasomal
inhibitor MG132 (lane 3). MG132 did not interfere with
Hec1/Nek2 interaction (Figure 3A, lane 5), nor did it impact
the ability of 32 to disrupt Hec1/Nek2 interaction (Figure 3A,
lane 4). Taken together, these results suggested that 32
disrupted the Hec1/Nek2 complex in the cells and that the
detached Nek2 might be degraded through the proteasomal
pathway, as reported by Fry et al.21,22
Hec1 plays a critical role in mitosis, and intervening in its
function leads to mitotic abnormalities.16,17 In MDA-MB-468
cells treated with 32, the percentage of mitotic abnormalities,
including chromosomal misalignment and formation of multi-
polar spindles, was measured by immunofluorescent staining
and microscopy (Table 5). The percentage of cells displaying
multipolar spindles increased in a dose-dependent manner:
16.9, 29.1, and 46.7% for cells treated with 32 at concentrations
of 10, 100, and 1000 nM, respectively. However, no significant
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Table 3. In Vitro Potency of 4-Aryl-N-(4-pyridylcarbonyl)-2-aminothiazole 13−15, 18, and 32 Bearing a C-4′ Alkoxy or Aryloxy,
C-2′ Methyl or Fluoro, and o-Hydro or Fluoro or Chloro at the Pyridyl Group
a
The IC50 values were averaged from two independent dose−response curves; variation was generally <15%.
a
Table 4. Pharmacokinetic Parameters (IV, PO) of Compounds 9a, 12, 17, 31, and 32
b
c
IV
PO
b
compd
MB231 IC50 (nM)
AUC (μM·h)
Vd (L/kg)
CL (mL/min/kg)
F%
9a
12
17
31
32
300
160
96.0
32.0
5.78
32.7
44.3
34.3
64.9
1.54
23.2
3.02
2.55
2.70
1.22
41.9
20.6
22.6
12.4
0.250
0.769
1.26
d
20.0
0.948
4.6 (22.7)
a
b
c
Carried out in Sprague−Dawley rats. IV dose: 2.0 mg/kg formulated with 5% DMSO, 10% cremophor, and 85% water. PO dose: 20 mg/kg
d
formulated with 1% methylcellulose in water. Formulated with 5% DMSO, 10% cremophor, and 85% water.
change in the percentage of cells with chromosomal misalign-
ment was observed. These data indicated that 32 induced
mitotic abnormalities in the same manner as 1 and 2, even at a
concentration as low as 10 nM (IC50 of 32 for MDA-MB-468:
40.0 nM, Table 3). Furthermore, reduced levels of
antiapoptotic proteins, including Bcl-2, Mcl-1, and XIAP,
were observed in HeLa cells treated with 32 for 24 and 48 h,
whereas increasing levels of apoptotic marker proteins,
including cleaved caspase-3 and PARP, were noted (Figure
4). Combined with the results from coimmunoprecipitation,
immunofluorescent staining, and western blotting, we propose
that antiproliferative compound 32 acted by disrupting the
Hec1/Nek2 interaction, which led to mitotic abnormalities and
eventually to the apoptosis of the cancer cells.
Spectrum and Selectivity. To further evaluate the
spectrum of activity for Hec1/Nek2 inhibition within distinct
cancer cells, the GI50 values of 32 were determined. The results
shown in Table 6 indicated that 32 was active toward various
cancer cells, including CML, lymphoma, cervical cancer, colon
cancer, breast cancer, and hepatoma, with GI50 values less than
1.0 μM (22.8−377 nM). The observed broad spectrum activity
of 32 toward various cancer cells was anticipated because Hec1
plays a critical role in mitosis. On the contrary, compound 32
was not active (GI50 > 9.0 μM, Table 6) on normal cells,
including WI-38 (human normal lung fibroblast), RPTEC
(renal proximal tubule epithelial), HUVEC (human umbilical
vein endothelial), and HAoSMC (human aortic smooth
muscle) cells. These results suggested that compound 32
selectively killed cancer cells over normal cells. The differences
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CHK2, Cdk1/cyclin B, Aurora A, Aurora B, mTOR, PI3Kα,
PI3Kβ, VEGFR2, PDGFR-β, FGFR1, EGFR, EGFR(T790M),
IGF-1R, B-Raf, B-Raf(V600E), C-Raf, FLT3, MET, and Kit
kinases (IC50 > 10 μM), despite sharing a similar 2-
aminothiazole structure with the known kinase inhibitor,
dasatinib.24 Finally, 32 was inactive in the [3H]astemizole
competitive binding assay for hERG liability screening,25,26 with
an IC50 greater than 10 μM. Thus, it is not likely to cause QT
prolongation and torsade de pointes27 that often cause the
withdrawal of marketed drugs and/or failure in clinical trials.
Considering its potency, pharmacokinetic properties, and
selectivity, we believe that compound 32 could be a good
lead for further optimization.
CONCLUSIONS
■
We explored 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold
4 as Hec1/Nek2 inhibitors. Among the derivatives of 4,
compound 32 showed low nanomolar antiproliferative activity,
good pharmacokinetic properties, and significant in vivo
antitumor activity while being less active toward normal cells,
kinases, and hERG. Compound 32 will serve as a good lead in
our search for a preclinical candidate targeting Hec1/Nek2
inhibition.
Figure 2. In vivo antitumor activity of compound 32 in nude mice
bearing MDA-MB-231 breast cancer xenografts. Compound 32 was
formulated with a vehicle of 5% DMSO, 10% cremophor, and 85%
water. The average tumor volumes were recorded from mice receiving
⧫
a 28 day, continuous once-a-day IV dose of the vehicle ( ) or 32 at 20
●
mg/kg ( ) as well as from mice receiving a twice-a-day PO dose of 32
EXPERIMENTAL SECTION
▲
at 150 mg/kg ( ). The T/C values for 32 from the IV and PO routes
were calculated to be 32 and 57% on day 28, respectively.
■
General Procedures. Reagents and starting materials were used as
purchased without further purification. Analytical thin-layer chroma-
tography (TLC) was performed on precoated plates (silica gel 60 F-
254) purchased from Merck Inc. Purification by gravity column
chromatography was conducted using Merck Reagents Silica Gel 60
1
(with a particle size of 0.063−0.200 mm, 70−230 mesh ASTM). H
NMR spectra were recorded on a Bruker (500 MHz) spectrometer
with CDCl3, CD3OD, or DMSO-d6 as solvent. Multiplicities are
abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; and
m, multiplet; J, coupling constant (hertz). 13C NMR spectra were
obtained on an Agilent 400-MR (100 MHz) NMR spectrometer by
use of CDCl3 or DMSO-d6 as solvent. 13C chemical shifts are
referenced to the center peak of CDCl3 (δ 77.0 ppm) or DMSO-d6 (δ
39.5 ppm). ESI−MS spectra were recorded with an Applied
Biosystems API 300 mass spectrometer. High-resolution mass spectra
were obtained by means of a LTQ Orbitrap XL mass spectrometer
(Thermo Fisher Scientific). The purities of the compounds for
biological evaluation were greater than 95%, as determined from
reversed-phase HPLC. Reference compound 216 and 1-mesityletha-
none (6)18 were prepared according to reported procedures and
showed consistent spectroscopic data.
2-Bromo-1-mesitylethanone (7). To a solution of 1-mesityle-
thanone (6, 1.18 g, 7.27 mmol) in EtOAc (50 mL) was added CuBr2
(3.25 g, 14.6 mmol, 2.0 equiv). The reaction mixture was heated at
reflux for 90 min. The solution was cooled, and the resultant solids
were filtered off and washed with EtOAc. The filtrate was concentrated
under reduced pressure to give 2-bromo-1-mesitylethanone (7, 1.67 g,
6.93 mmol) as a yellow oil in 95% yield: 1H NMR (CDCl3) δ 6.87 (s,
2H), 4.27 (s, 2H), 2.31 (s, 3H), 2.22 (s, 6H); 13C NMR (CDCl3) δ
200.21, 139.60, 133.51, 128.98, 128.66, 36.67, 21.08, 19.34; ESI−MS:
m/z 241.0 (M + H)+.
Figure 3. Disruption of Hec1/Nek2 interaction by compound 32 in
the cells. (A) Coimmunoprecipitation analysis of K562 cells treated
with DMSO (lane 2), 32 (lane 3), 32 with MG132 (lane 4), and
MG132 (lane 5) for 16 h. The concentrations of 32 and MG132 were
100 and 500 nM, respectively. (B) Dose-dependent western blot of
Hec1 and Nek2 in K562 cells treated with DMSO or 32 at 10, 100,
and 1000 nM (lanes 1−4) for 24 h. Actin was used as a loading
control. (C) Western blot of Hec1 and Nek2 in MDA-MB-468 cells
treated with 32 (lane 2), 32 with MG132 (lane 3), and MG132 (lane
4). Actin was used as a loading control.
4-Mesitylthiazol-2-amine (8). Compound 7 (2.43 g, 10.1 mmol,
1.0 equiv) and thiourea (0.810 g, 10.6 mmol, 1.05 equiv) were
dissolved in 95% ethanol (20 mL). The reaction mixture was heated at
reflux for 2.0 h. The solution was concentrated under reduced
pressure, and the residue was recrystallized from 2-propanol to give 4-
mesitylthiazol-2-amine (8, 1.99 g, 9.12 mmol) as white solids in 90%
in GI50 values between cancer and normal cells gave further
proof of the good in vivo tolerability of 32 and implied that it
has a good therapeutic window.
For further assessment of the selectivity of 32, radiometric
assays23 were used to address its inhibitory activity against 21
kinases. Compound 32 was not active against Nek2, CHK1,
1
yield: H NMR (CD3OD) δ 7.00 (s, 2H), 6.67 (s, 1H), 2.31 (s, 3H),
2.19 (s, 6H); 13C NMR (CDCl3) δ 167.66, 137.55, 137.34, 131.96,
128.50, 128.06, 105.12, 21.08, 20.25; ESI−MS: m/z 219.1 (M + H)+.
1-(4-Chloro-2,6-dimethylphenyl)ethanone (22). Anhydrous
CuCl2 (98.9 g, 0.736 mol, 1.2 equiv) was mixed with t-BuONO
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Table 5. Immunofluorescent Staining (Microscopy, 1000×), Percentage of Chromosomal Misalignment, and Formation of
Multipolar Spindles in Mitotic MDA-MB-468 Cells Treated with 32 at Various Concentrations for 48 h
a
DMSO (control).
(85.6 g, 0.830 mol, 1.4 equiv) in CH3CN (1.02 L). The solution was
cooled to 0 °C, and 1-(4-amino-2,6-dimethylphenyl)ethanone (21,
100 g, 0.613 mol, 1.0 equiv) was slowly added over a period of 5.0 min.
After the addition was completed, the reaction mixture was warmed to
room temperature and poured into an aqueous hydrochloric acid
solution (20%, 1.0 L). The solution was extracted with EtOAc (800
mL), and the organic layer was separated, washed with H2O (1.0 L),
dried over MgSO4(s), and concentrated under reduced pressure. The
liquid was distilled to give 1-(4-chloro-2,6-dimethylphenyl)ethanone
(22, 85.0 g, 0.465 mol) as a yellow oil in 76% yield: 1H NMR (CDCl3)
δ 7.02 (s, 2H), 2.45 (s, 3H), 2.22 (s, 6H); 13C NMR (CDCl3) δ
207.29, 140.87, 134.28, 133.92, 127.66, 77.33, 77.02, 76.70, 32.03,
19.07, 18.98; ESI−MS: m/z 183.0 (M + H)+.
General Procedure for the Synthesis of 4-Aryloxy-2,6-
dimethylacetophenones 23a−i. To a solution of 1-(4-chloro-2,6-
dimethylphenyl)ethanone (22, 1.0 equiv), K3PO4 (2.0 equiv), and
various aryl alcohols (1.2 equiv) in toluene were added t-BuXPhos
(0.030 equiv) and Pd(OAc)2 (0.050 equiv). The reaction mixture was
heated at 100 °C for 2.0 h under N2. The solution was cooled to room
temperature and filtered through a small pad of Celite. The cake was
washed with EtOAc, and the combined filtrate was concentrated under
reduced pressure. The residue was purified by gravity column
chromatography on silica gel (mixture of EtOAc and hexanes as
Figure 4. Western blot analysis of cleaved caspase-3, cleaved PARP,
eluent) to give corresponding 4-aryloxy-2,6-dimethylacetophenones
23a−i in 19−86% yields.
Bcl-2, Mcl-1, and XIAP in HeLa cells treated with DMSO, compound
32 (1.0 μM), or paclitaxel (100 nM) for 24 and 48 h. Actin was used
as a loading control.
1-(2,6-Dimethyl-4-phenoxyphenyl)ethanone (23a). Yield:
1
68%. H NMR (CDCl3) δ 7.33−7.37 (m, 2H), 7.12 (t, J = 7.5 Hz,
1H), 7.00 (d, J = 7.5 Hz, 2H), 6.65 (s, 2H), 2.48 (s, 3H), 2.22 (s, 6H);
Table 6. GI50 Values of Compound 32 toward Various Cancer and Normal Cells
a
a
cell
K562
origin
CML
GI50 (nM)
cell
Huh7
origin
GI50
56.2
29.6
22.8
34.1
35.4
39.8
29.6
377
hepatoma
hepatoma
hepatoma
hepatoma
normal
79.6 nM
79.6 nM
191 nM
68.3 nM
>9.0 μM
>9.0 μM
>9.0 μM
>9.0 μM
U937
lymphoma
cervical
colon
PLC/PRF/5
HepG2
HeLa
HCT116
COLO205
MB231
MB468
T47D
Hep3B
colon
WI-38
breast
RPTEC
HUVEC
HAoSMC
normal
breast
normal
breast
normal
a
The GI50 values were averaged from two independent dose−response curves; variation was generally <15%.
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13C NMR (CDCl3) δ 207.66, 157.21, 156.72, 137.74, 134.70, 129.79,
123.53, 119.20, 117.71, 32.25, 19.37; ESI−MS: m/z 241.1 (M + H)+.
1-[2,6-Dimethyl-4-(p-tolyloxy)phenyl]ethanone (23b). Yield:
4-(2,6-Difluoro-4-methoxyphenyl)thiazol-2-amine (11b).
1
Yield: 57%. H NMR (CDCl3) δ 6.68 (s, 1H), 6.51 (d, J = 10 Hz,
2H), 5.07 (brs, 2H), 3.81 (s, 3H); 13C NMR (DMSO-d6) δ 168.22,
160.93 (dd, J = 245.7, 10.4 Hz), 160.31 (t, J = 14.3 Hz), 138.50,
107.35, 106.30 (t, J = 18.7 Hz), 98.70 (dd, J = 21.4, 7.9 Hz), 56.30;
ESI−MS: m/z 243.2 (M + H)+.
1
46%. H NMR (CDCl3) δ 7.15 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4
Hz, 2H), 6.62 (s, 2H), 2.66 (s, 3H), 2.35 (s, 3H), 2.21 (s, 6H); 13C
NMR (CDCl3) δ 207.99, 157.75, 154.12, 137.29, 134.61, 133.23,
130.26, 119.40, 117.14, 32.31, 20.71, 19.39; ESI−MS: m/z 255.3 (M +
H)+.
4-(4-Ethoxy-2,6-dimethylphenyl)thiazol-2-amine (11c).
1
Yield: 72%. H NMR (DMSO-d6) δ 6.84 (brs, 2H), 6.60 (s, 2H),
6.27 (s, 1H), 3.99 (q, J = 6.8 Hz, 2H), 2.06 (s, 6H), 1.31 (t, J = 6.8 Hz,
3H); 13C NMR (DMSO-d6) δ 168.07, 157.83, 149.39, 138.44, 129.14,
113.31, 104.18, 63.14, 20.83, 15.16; ESI−MS: m/z 249.1 (M + H)+.
4-(4-Isopropoxy-2,6-dimethylphenyl)thiazol-2-amine (11d).
Yield: 72%. 1H NMR (CDCl3) δ 6.60 (s, 2H), 6.26 (s, 1H), 4.97 (bs,
2H), 4.51−4.56 (m, 1H), 2.13 (s, 6H), 1.32 (d, J = 6.1 Hz, 6H); 13C
NMR (DMSO-d6) δ 168.71, 158.73, 138.91, 126.33, 113.57, 104.86,
74.04, 28.18, 20.67, 19.48; ESI−MS: m/z 263.2 (M + H)+.
1-[4-(4-Ethylphenoxy)-2,6-dimethylphenyl]ethanone (23c).
Yield: 82%. H NMR (CDCl3) δ 7.17 (d, J = 8.5 Hz, 2H), 6.93 (d,
1
J = 8.5 Hz, 2H), 6.63 (s, 2H), 2.64 (q, J = 7.5 Hz, 2H), 2.47 (s, 3H),
2.21 (s, 6H), 1.25 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3) δ 207.92,
157.69, 154.34, 139.58, 137.35, 134.62, 129.06, 119.33, 117.26, 32.31,
28.16, 19.40, 15.68; ESI−MS: m/z 269.2 (M + H)+.
1-[4-(3,5-Dimethylphenoxy)-2,6-dimethylphenyl]ethanone
1
(23d). Yield: 86%. H NMR (CDCl3) δ 6.76 (s, 1H), 6.63 (s, 2H),
6.62 (s, 2H), 2.48 (s, 3H), 2.29 (s, 6H), 2.22 (s, 6H); 13C NMR
(CDCl3) δ 208.03, 157.39, 156.67, 139.59, 137.48, 134.58, 125.23,
117.72, 116.85, 32.28, 21.26, 19.37; ESI−MS: m/z 269.1 (M + H)+.
1-[4-(4-Fluorophenoxy)-2,6-dimethylphenyl]ethanone
4-(4-Isobutoxy-2,6-dimethylphenyl)thiazol-2-amine (11e).
1
Yield: 82%. H NMR (CDCl3) δ 6.61 (s, 2H), 6.24 (s, 1H), 3.70
(d, J = 6.5 Hz, 2H), 2.15 (s, 6H), 2.05−2.09 (m, 1H), 1.01 (d, J = 6.7
Hz, 6H); 13C NMR (CDCl3) δ 167.55, 158.68, 148.83, 138.92, 127.16,
113.27, 105.30, 74.27, 28.28, 20.58, 19.28; ESI−MS: m/z 277.0 (M +
H)+.
1
(23e). Yield: 68%. H NMR (CDCl3) δ 7.01−7.05 (m, 2H), 6.70−
6.96 (m, 2H), 6.60 (s, 2H), 2.47 (s, 3H), 2.22 (s, 6H); 13C NMR
(CDCl3) δ 207.68, 158.89 (d, J = 262.4 Hz), 157.53, 152.33, 137.58,
134.76, 120.84 (d, J = 8.3 Hz), 117.09, 116.32 (d, J = 23.2 Hz), 32.28,
19.39; ESI−MS: m/z 259.0 (M + H)+.
4-[4-(Cyclopentyloxy)-2,6-dimethylphenyl]thiazol-2-amine
1
(11f). Yield: 74%. H NMR (CDCl3) δ 6.58 (s, 2H), 6.24 (s, 1H),
4.73−4.77 (m, 1H), 2.13 (s, 6H), 1.78−1.88 (m, 6H), 1.59−1.62 (m,
2H); 13C NMR (DMSO-d6) δ 168.17, 157.03, 148.47, 138.48, 128.35,
114.35, 104.27, 78.69, 32.78, 24.04, 20.80; ESI−MS: m/z 289.1 (M +
H)+.
4-(2,6-Dimethyl-4-phenoxyphenyl)thiazol-2-amine (24a).
Yield: 59%. 1H NMR (CDCl3) δ 7.31−7.35 (m, 2H), 7.10−7.26
(m, 3H), 6.72 (s, 2H), 6.30 (s, 1H), 5.18 (bs, 2H), 2.14 (s, 6H); 13C
NMR (DMSO-d6) δ 168.49, 157.14, 156.11, 147.70, 139.39, 131.47,
130.39, 123.72, 119.07, 117.51, 104.63, 20.66; ESI−MS: m/z 297.1 (M
+ H)+.
4-[2,6-Dimethyl-4-(p-tolyloxy)phenyl]thiazol-2-amine (24b).
Yield: 53%. 1H NMR (DMSO-d6) δ 7.23 (d, J = 8.3 Hz, 2H), 6.94 (d, J
= 8.3 Hz, 2H), 6.75 (br, 3H), 2.36 (s, 3H), 2.12 (s, 6H); 13C NMR
(DMSO-d6) δ 170.00, 158.66, 153.83, 140.50, 135.96, 133.66, 130.99,
123.74, 119.86, 116.90, 106.53, 20.77, 20.39; ESI−MS: m/z 311.2 (M
+ H)+.
4-[4-(4-Ethylphenoxy)-2,6-dimethylphenyl]thiazol-2-amine
(24c). Yield: 88%. 1H NMR (CDCl3) δ 7.18 (d, J = 8.0 Hz, 2H), 6.95
(d, J = 8.0 Hz, 2H), 6.71 (s, 2H), 6.29 (s, 1H), 5.45 (bs, 2H), 2.64 (q, J
= 7.6 Hz, 2H), 2.14 (s, 6H), 1.25 (t, J = 7.6 Hz, 3H); 13C NMR
(CDCl3) δ 167.56, 157.21, 154.80, 147.77, 139.42, 139.19, 134.63,
128.96, 119.07, 117.13, 105.57, 28.14, 20.48, 15.70; ESI−MS: m/z
325.0 (M + H)+.
1-{2,6-Dimethyl-4-[4-(trifluoromethyl)phenoxy]phenyl}-
ethanone (23f). Yield: 19%. 1H NMR (CDCl3) δ 7.58 (d, J = 8.5 Hz,
2H), 7.04 (d, J = 8.5 Hz, 2H), 6.70 (s, 2H), 2.50 (s, 3H), 2.25 (s, 6H);
13C NMR (CDCl3) δ 207.66, 160.05, 155.58, 138.89, 135.01, 127.11
(q, J = 3.7 Hz), 126.60 (q, J = 215 Hz), 125.06 (q, J = 32.6 Hz),
118.79, 118.13, 32.18, 19.28; ESI−MS: m/z 309.0 (M + H)+.
1-[4-(4-Methoxyphenoxy)-2,6-dimethylphenyl]ethanone
(23g). Yield: 75%. 1H NMR (CDCl3) δ 6.96 (d, J = 9.2 Hz, 2H), 6.88
(d, J = 9.2 Hz, 2H), 6.57 (s, 2H), 3.81 (s, 3H), 2.46 (s, 3H), 2.20 (s,
6H); 13C NMR (DMSO-d6) δ 207.63, 158.21, 156.22, 149.32, 137.50,
134.85, 121.42, 116.42, 115.52, 55.80, 32.58, 19.33; ESI−MS: m/z
271.1 (M + H)+.
1-[4-(3-Methoxyphenoxy)-2,6-dimethylphenyl]ethanone
1
(23h). Yield: 73%. H NMR (CDCl3) δ 7.23−7.25 (m, 1H), 6.66−
6.68 (m, 3H), 6.56−6.57 (m, 2H), 3.79 (s, 3H), 2.48 (s, 3H), 2.22 (s,
6H); 13C NMR (CDCl3) δ 207.89, 160.95, 157.91, 156.94, 137.81,
134.66, 130.15, 117.86, 111.26, 109.11, 105.18, 55.34, 32.27, 19.36;
ESI−MS: m/z 271.0 (M + H)+.
1-[4-(Benzo[d][1,3]dioxol-5-yloxy)-2,6-dimethylphenyl]-
ethanone (23i). Yield: 62%. 1H NMR (CDCl3) δ 6.77 (d, J = 8.5 Hz,
1H), 6.59 (s, 2H), 6.56 (s, 1H), 6.46−6.49 (m, 1H), 5.98 (s, 2H), 2.46
(s, 3H), 2.21 (s, 6H); 13C NMR (CDCl3) δ 207.90, 158.10, 150.79,
148.36, 143.96, 137.24, 134.64, 116.67, 112.24, 108.28, 102.35, 101.54,
32.30, 19.41; ESI−MS: m/z 285.1 (M + H)+.
4-[4-(3,5-Dimethylphenoxy)-2,6-dimethylphenyl]thiazol-2-
amine (24d). Yield: 59%. 1H NMR (CDCl3) δ 6.76 (s, 1H), 6.68 (s,
2H), 6.64 (s, 2H), 6.26 (s, 1H), 2.29 (s, 6H), 2.16 (s, 6H); 13C NMR
(DMSO-d6) δ 168.77, 156.94, 156.77, 139.73, 139.51, 129.64, 125.50,
119.21, 117.44, 116.89, 104.99, 21.31, 20.59; ESI−MS: m/z 325.1 (M
+ H)+.
General Procedure for the Synthesis of 4-Aryl-2-amino-
thiazoles 11a−f and 24a−i. To a CH3CN solution of acetylarenes
10a−f or 23a−i (1.0 equiv) was added TBABr3 (1.0 equiv). The
reaction mixture was stirred at room temperature for 80 min. The
solution was concentrated under reduced pressure, diluted with H2O,
and extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous MgSO4(s), and concentrated under reduced
pressure to give the brominated acetylarene. Thiourea (1.0 equiv) was
added to the brominated acetylarene in EtOH, and the reaction
mixture was heated at reflux for 60 min. After the reaction was
completed, the solution was concentrated, and water and saturated
aqueous Na2CO3 were added. The resultant precipitate was filtered
and recrystallized in toluene (50 mL). The solids were collected and
dried under vacuum to give the desired 4-aryl-2-aminothiazoles 11a−f
or 24a−i in 53−96% yields.
4-[4-(4-Fluorophenoxy)-2,6-dimethylphenyl]thiazol-2-
1
amine (24e). Yield: 84%. H NMR (CDCl3) δ 6.97−7.05 (m, 4H),
6.66 (s, 2H), 6.28 (s, 1H), 5.95 (bs, 2H), 2.14 (s, 6H); 13C NMR
(DMSO-d6) δ 168.96, 158.69 (d, J = 238 Hz), 157.18, 152.80 (d, J =
2.3 Hz), 143.54, 139.84, 128.90, 121.26 (d, J = 8.5 Hz), 117.04 (d, J =
23.2 Hz), 117.00, 105.27, 20.54; ESI−MS: m/z 315.1 (M + H)+.
4-{2,6-Dimethyl-4-[4-(trifluoromethyl)phenoxy]phenyl}-
thiazol-2-amine (24f). Yield: 63%. 1H NMR (CDCl3) δ 7.56 (d, J =
8.5 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 6.76 (s, 2H), 6.32 (s, 1H), 5.03
(brs, 2H), 2.17 (s, 6H); 13C NMR (DMSO-d6) δ 168.48, 160.92,
154.28, 148.40, 139.73, 133.38, 127.68, 124.69 (q, J = 207 Hz), 123.66
(q, J = 32.1 Hz), 118.68, 118.20, 104.51, 20.55; ESI−MS: m/z 365.1
(M + H)+.
4-(4-Methoxy-2,6-dimethylphenyl)thiazol-2-amine (11a).
1
Yield: 66%. H NMR (CDCl3) δ 6.61 (s, 2H), 6.27 (s, 1H), 4.91
4-[4-(4-Methoxyphenoxy)-2,6-dimethylphenyl]thiazol-2-
(bs, 2H), 3.79 (s, 3H), 2.15 (s, 6H); 13C NMR (DMSO-d6) δ 169.91,
160.36, 139.83, 136.30, 121.33, 113.42, 106.29, 55.69, 20.49; ESI−MS:
m/z 235.1 (M + H)+.
1
amine (24g). Yield: 68%. H NMR (CDCl3) δ 6.98 (d, J = 9.0 Hz,
2H), 6.88 (d, J = 9.0 Hz, 2H), 6.64 (s, 2H), 6.27 (s, 1H), 5.40 (bs,
2H), 3.81 (s, 3H), 2.13 (s, 6H); 13C NMR (DMSO-d6) δ 168.36,
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157.47, 155.99, 149.83, 147.77, 139.17, 130.62, 121.15, 116.19, 115.46,
104.59, 55.80, 20.67; ESI−MS: m/z 327.1 (M + H)+.
151.20, 150.84, 139.56, 122.23, 115.26, 105.48 (t, J = 19.0 Hz), 99.02
(dd, J = 21.2, 7.9 Hz), 56.62; ESI−MS: m/z 348.1 (M + H)+.
2-Fluoro-N-[4-(4-methoxy-2,6-dimethylphenyl)thiazol-2-yl]-
isonicotinamide (14). Yield: 68%. 1H NMR (CDCl3) δ 8.37 (d, J =
5.2 Hz, 1H), 7.72 (brs, 1H), 7.42 (d, J = 5.2 Hz, 1H), 6.84 (s, 1H),
6.48 (s, 2H), 3.78 (s, 3H), 2.03 (s, 6H); 13C NMR (CDCl3) δ 163.87
(d, J = 242.2 Hz), 162.69, 159.45, 159.07, 147.96 (d, J = 14.5 Hz),
147.58, 144.13 (d, J = 7.5 Hz), 138.31, 125.41, 118.97 (d, J = 4.4 Hz),
112.79, 112.38, 107.98 (d, J = 39.1 Hz), 54.88, 20.34; ESI−MS: m/z
358.0 (M + H)+; HRMS calcd for C18H17FN3O2S (M+ + H),
358.1015; found, 358.1020.
2-Chloro-N-[4-(4-methoxy-2,6-dimethylphenyl)thiazol-2-yl]-
isonicotinamide (15). Yield: 63%. 1H NMR (CDCl3) δ 8.60 (d, J =
5.1 Hz, 1H), 7.95 (s, 1H), 7.92 (d, J = 5.1 Hz, 1H), 6.87 (s, 1H), 6.58
(s, 2H), 3.81 (s, 3H), 2.11 (s, 6H); 13C NMR (CDCl3) δ 162.82,
159.61, 159.16, 152.17, 149.80, 147.41, 141.68, 138.35, 125.32, 122.34,
119.77, 112.78, 112.30, 54.98, 20.45; ESI−MS: m/z 373.9 (M + H)+.
N-[4-(4-Ethoxy-2,6-dimethylphenyl)thiazol-2-yl]-
isonicotinamide (16). Yield: 88%. 1H NMR (CDCl3) δ 8.70 (d, J =
6.0 Hz, 2H), 7.58 (d, J = 6.0 Hz, 2H), 6.78 (s, 1H), 6.37 (s, 2H), 3.95
(q, J = 7.0 Hz, 2H), 1.94 (s, 6H), 1.41 (t, J = 7.0 Hz, 3H); 13C NMR
(CDCl3) δ 163.76, 159.09, 158.34, 150.10, 147.98, 138.60, 138.18,
125.68, 121.00, 113.34, 112.13, 63.06, 20.45, 14.88; ESI−MS: m/z
353.6 (M + H)+.
N-[4-(4-Isopropoxy-2,6-dimethylphenyl)thiazol-2-yl]-
isonicotinamide (17). Yield: 80%. 1H NMR (CDCl3) δ 8.67 (d, J =
6.0 Hz, 2H), 7.55 (d, J = 6.0 Hz, 2H), 6.77 (s, 1H), 6.30 (s, 2H),
4.41−4.45 (m, 1H), 1.90 (s, 6H), 1.32 (d, J = 6.0 Hz, 6H); 13C NMR
(CDCl3) δ 163.75, 159.05, 157.36, 150.09, 148.01, 138.62, 138.20,
125.54, 121.02, 114.35, 112.15, 69.27, 22.13, 20.49; ESI−MS: m/z
368.1 (M + H)+.
2-Fluoro-N-[4-(4-isopropoxy-2,6-dimethylphenyl)thiazol-2-
yl]isonicotinamide (18). Yield: 83%. 1H NMR (CDCl3) δ 8.40 (d, J
= 5.1 Hz, 1H), 7.78 (s, 1H), 7.50 (d, J = 5.1 Hz, 1H), 6.84 (s, 1H),
6.53 (s, 2H), 4.52−4.56 (m, 1H), 2.06 (s, 6H), 1.33 (d, J = 6.0 Hz,
6H); 13C NMR (CDCl3) δ 163.89 (d, J = 241.3 Hz), 162.72, 159.47,
157.52, 147.91 (d, J = 14.5 Hz), 147.61, 144.13 (d, J = 7.5 Hz), 138.29,
125.05, 118.99 (d, J = 4.4 Hz), 114.21, 112.29, 108.06 (d, J = 39.1 Hz),
69.24, 22.04, 20.39; ESI−MS: m/z 385.8 (M + H)+.
N-[4-(4-Isobutoxy-2,6-dimethylphenyl)thiazol-2-yl]-
isonicotinamide (19). Yield: 99%. 1H NMR (CDCl3) δ 8.68 (d, J =
5.7 Hz, 2H), 7.56 (d, J = 5.7 Hz, 2H), 6.77 (s, 1H), 6.33 (s, 2H), 3.62
(d, J = 6.5 Hz, 2H), 2.05−2.12 (m, 1H), 1.91 (s, 6H), 1.05 (d, J = 6.7
Hz, 6H); 13C NMR (CDCl3) δ 163.78, 159.16, 158.65, 150.07, 147.95,
138.59, 138.16, 125.52, 121.04, 113.30, 112.08, 73.99, 28.29, 20.42,
19.29; ESI−MS: m/z 382.1 (M + H)+.
N-{4-[4-(Cyclopentyloxy)-2,6-dimethylphenyl]thiazol-2-yl}-
isonicotinamide (20). Yield: 62%. 1H NMR (CDCl3) δ 8.71 (d, J =
5.8 Hz, 2H), 7.60 (d, J = 5.8 Hz, 2H), 6.78 (s, 1H), 6.37 (s, 2H),
4.65−4.69 (m, 1H), 1.95 (s, 6H), 1.52−1.94 (m, 8H); 13C NMR
(CDCl3) δ 163.71, 159.06, 157.61, 150.13, 148.01, 138.62, 138.14,
125.32, 121.04, 114.26, 112.11, 78.77, 32.86, 23.94, 20.51; ESI−MS:
m/z 394.1 (M + H)+.
N-[4-(2,6-Dimethyl-4-phenoxyphenyl)thiazol-2-yl]-
isonicotinamide (25). Yield: 30%. 1H NMR (DMSO-d6) δ 8.81 (d, J
= 5.5 Hz, 2H), 7.99 (d, J = 5.5 Hz, 2H), 7.39−7.42 (m, 2H), 7.03−
7.19 (m, 4H), 6.78 (s, 2H), 2.07 (s, 6H); 13C NMR (CDCl3) δ 163.74,
159.08, 156.93, 156.43, 150.33, 147.66, 138.75, 129.78, 128.31, 123.56,
121.04, 119.32, 117.17, 112.40, 77.36, 77.05, 76.73, 20.43; ESI−MS:
m/z 401.8 (M + H)+.
N-{4-[2,6-Dimethyl-4-(p-tolyloxy)phenyl]thiazol-2-yl}-
isonicotinamide (26). Yield: 89%. 1H NMR (DMSO-d6) δ 8.80 (d, J
= 5.5 Hz, 2H), 7.99 (d, J = 5.5 Hz, 2H), 7.18−7.22 (m, 3H), 6.95 (d, J
= 8.4 Hz, 2H), 6.73 (s, 2H), 2.30 (s, 3H), 2.06 (s, 6H); 13C NMR
(CDCl3) δ 163.68, 159.02, 157.50, 153.91, 150.37, 147.72, 138.76,
138.70, 133.26, 130.28, 127.91, 121.04, 119.52, 116.66, 112.32, 20.74,
20.43; ESI−MS: m/z 413.9 (M − H)−.
4-[4-(3-Methoxyphenoxy)-2,6-dimethylphenyl]thiazol-2-
1
amine (24h). Yield: 65%. H NMR (CDCl3) δ 7.20−7.23 (m, 1H),
6.64 (s, 2H), 6.60−6.63 (m, 1H), 6.45−6.50 (m, 2H), 6.30 (s, 1H),
3.81 (s, 3H), 1.94 (s, 6H); 13C NMR (DMSO-d6) δ 168.43, 161.10,
158.41, 155.79, 148.74, 139.27, 132.14, 130.78, 117.60, 110.99, 109.21,
105.17, 104.45, 55.57, 20.66; ESI−MS: m/z 327.0 (M + H)+.
4-[4-(Benzo[d][1,3]dioxol-5-yloxy)-2,6-dimethylphenyl]-
thiazol-2-amine (24i). Yield: 96%. 1H NMR (CDCl3) δ 6.75 (d, J =
8.5 Hz, 1H), 6.66 (s, 2H), 6.57−6.59 (m, 1H), 6.48−6.51 (m, 1H),
6.28 (s, 1H), 5.98 (s, 2H), 5.05 (brs, 2H), 2.13 (s, 6H); 13C NMR
(DMSO-d6) δ 168.19, 157.06, 151.16, 148.57, 143.94, 139.09, 131.46,
116.56, 116.32, 112.22, 108.84, 104.45, 102.45, 101.98, 20.69; ESI−
MS: m/z 341.0 (M + H)+.
General Procedure for the Synthesis of 4-Aryl-N-acyl-2-
aminothiazoles 9a−e, 12−20, and 25−34. To a solution of 4-aryl-
2-aminothiazoles 8 or 11a−f or 24a−i (1.0 equiv) in anhydrous
CH2Cl2 was added DMAP (3.0 equiv) followed by various acyl
chlorides (1.5 equiv) under N2. The reaction mixture was stirred at
room temperature for 12 h. The solution was concentrated under
reduced pressure, and hot water was added. After cooling, the resultant
precipitate was filtered, dried under vacuum, and purified by gravity
column chromatography on silica gel (mixture of EtOAc and hexanes
as eluent) to give the corresponding 4-aryl-N-acyl-2-aminothiazoles
9a−e, 12−20, and 25−34 in 17−99% yields.
1
N-(4-Mesitylthiazol-2-yl)isonicotinamide (9a). Yield: 77%. H
NMR (CD3OD) δ 8.72 (d, J = 6.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 2H),
6.80 (s, 1H), 6.65 (s, 2H), 2.20 (s, 3H), 1.94 (s, 6H); 13C NMR
(CDCl3) δ 163.75, 159.23, 149.91, 148.11, 138.48, 137.76, 136.55,
130.48, 128.24, 120.94, 112.02, 77.34, 77.02, 76.70, 20.83, 20.11; ESI−
MS: m/z 324.0 (M + H)+; HRMS calcd for C18H18N3OS (M+ + H),
324.1168; found, 324.1165.
N-(4-Mesitylthiazol-2-yl)-2-(pyridin-4-yl)acetamide (9b).
1
Yield: 65%. H NMR (DMSO-d6) δ 8.53 (d, J = 5.8 Hz, 2H), 7.36
(d, J = 5.8 Hz, 2H), 6.99 (s, 1H), 6.91 (s, 2H), 3.84 (s, 2H), 2.25 (s,
3H), 2.02 (s, 6H); 13C NMR (CDCl3) δ 167.88, 158.87, 149.84,
147.76, 142.34, 138.39, 137.39, 131.58, 128.70, 124.63, 111.72, 77.30,
76.98, 76.67, 40.34, 21.00, 20.32; ESI−MS: m/z 338.1 (M + H)+.
4-Cyano-N-(4-mesitylthiazol-2-yl)benzamide (9c). Yield: 67%.
1H NMR (DMSO-d6) δ 8.23 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz,
2H), 7.09 (s, 1H), 6.92 (s, 2H), 2.26 (s, 3H), 2.05 (s, 6H); 13C NMR
(CDCl3) δ 163.58, 159.72, 146.46, 138.35, 136.74, 135.00, 132.03,
129.31, 128.39, 128.30, 117.75, 116.04, 111.89, 20.94, 20.23; ESI−MS:
m/z 348.0 (M + H)+.
4-[(4-Mesitylthiazol-2-yl)carbamoyl]pyridine 1-Oxide (9d).
1
Yield: 75%. H NMR (CDCl3) δ 8.16 (d, J = 6.6 Hz, 2H), 7.79 (d,
J = 6.6 Hz, 2H), 6.83 (s, 1H), 6.80 (s, 2H), 2.23 (s, 3H), 2.01 (s, 6H);
13C NMR (CDCl3) δ 161.95, 159.63, 147.51, 138.82, 138.14, 136.85,
130.70, 128.58, 128.08, 124.50, 112.03, 77.35, 77.03, 76.71, 20.88,
20.06; 13C NMR (CDCl3) δ 161.95, 159.63, 147.51, 138.82, 138.14,
136.85, 130.70, 128.58, 128.08, 124.50, 112.03, 77.35, 77.03, 76.71,
20.88, 20.06; ESI−MS: m/z 340.1 (M + H)+.
2-Chloro-N-(4-mesitylthiazol-2-yl)isonicotinamide (9e).
1
Yield: 87%. H NMR (DMSO-d6) δ 8.50 (d, J = 5.1 Hz, 1H), 7.74
(d, J = 5.1 Hz, 1H), 7.62 (s, 1H), 6.83 (s, 1H), 6.72 (s, 2H), 2.26 (s,
3H), 1.97 (s, 6H); 13C NMR (CDCl3) δ 162.76, 159.58, 152.25,
149.59, 147.67, 141.60, 138.29, 136.62, 130.02, 128.23, 122.38, 119.65,
112.18, 20.98, 20.12; ESI−MS: m/z 357.9 (M + H)+.
N-[4-(4-Methoxy-2,6-dimethylphenyl)thiazol-2-yl]-
1
isonicotinamide (12). Yield: 69%. H NMR (CDCl3) δ 8.70 (d, J =
6.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 2H), 6.81 (s, 1H), 6.36 (s, 2H), 3.76
(s, 3H), 1.92 (s, 6H); 13C NMR (CDCl3) δ 163.88, 159.40, 158.89,
149.86, 147.65, 138.80, 138.23, 125.73, 121.14, 112.81, 112.12, 54.92,
20.43; ESI−MS: m/z 340.0 (M + H)+.
N-[4-(2,6-Difluoro-4-methoxyphenyl)thiazol-2-yl]-
isonicotinamide (13). Yield: 49%. 1H NMR (DMSO-d6) δ 13.13 (s,
1H), 8.81 (d, J = 5.9 Hz, 2H), 8.00 (d, J = 5.9 Hz, 2H), 7.46 (s, 1H),
6.87 (d, J = 10 Hz, 2H), 3.83 (s, 3H); 13C NMR (CDCl3) δ 164.39,
160.99 (t, J = 14.3 Hz), 160.89 (dd, J = 245.7, 10.2 Hz), 158.03,
N-{4-[4-(4-Ethylphenoxy)-2,6-dimethylphenyl]thiazol-2-yl}-
isonicotinamide (27). Yield: 91%. 1H NMR (CDCl3) δ 8.78 (d, J =
6.0 Hz, 2H), 7.67 (d, J = 6.0 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.93
(d, J = 8.4 Hz, 2H), 6.83 (s, 1H), 6.56 (s, 2H), 2.66 (q, J = 7.6 Hz,
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2H), 1.98 (s, 6H), 1.26 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3) δ
163.72, 159.02, 157.42, 154.04, 150.32, 147.72, 139.63, 138.76, 138.66,
129.07, 127.91, 121.04, 119.46, 116.72, 112.34, 28.17, 20.43, 15.67;
ESI−MS: m/z 429.6 (M + H)+.
incubated for another 3 h. Cell viability was determined by measuring
absorbance at 490 nm using an EMax microplate reader (Molecular
Devices). Data were processed and analyzed using GraphPad Prism,
version 4.
Pharmacokinetics in Rat. The pharmacokinetic properties of
compounds 9a, 12, 17, 31, and 32 were determined in male Sprague−
Dawley rats (n = 3). All procedures involving animals were performed
according to protocols approved by the IACUC in the Development
Center for Biotechnology. Compounds were formulated in an aqueous
solution containing 5% DMSO and 10% Cremophor for IV dosing at
2.0 mg/kg and in an aqueous 1% methylcellulose solution for PO
dosing at 20 mg/kg. After dosing, blood samples were taken at regular
time intervals over a period of 24 h from a preimplanted catheter, and
plasma was separated by centrifugation at 4000 rpm at 4 °C. Plasma
was subjected to deproteination by acetonitrile treatment, and the
concentration of the compounds was determined by LC−MS/MS.
Pharmacokinetic parameters were calculated using noncompartmental
analysis methods in WinNonlin software (version 5.2.1, Pharsight, CA,
USA).
N-{4-[4-(3,5-Dimethylphenoxy)-2,6-dimethylphenyl]thiazol-
2-yl}isonicotinamide (28). Yield: 94%. 1H NMR (CDCl3) δ 8.81 (d,
J = 5.4 Hz, 2H), 7.78 (d, J = 5.4 Hz, 2H), 6.84 (s, 1H), 6.78 (s, 1H),
6.64 (s, 2H), 6.61 (s, 2H), 2.31 (s, 6H), 2.02 (s, 6H); 13C NMR
(CDCl3) δ 163.72, 159.29, 157.32, 156.38, 150.03, 147.18, 139.61,
139.32, 138.85, 127.86, 125.36, 121.32, 117.19, 117.05, 112.16, 21.31,
20.45; ESI−MS: m/z 429.8 (M + H)+.
N-{4-[4-(4-Fluorophenoxy)-2,6-dimethylphenyl]thiazol-2-yl}-
1
isonicotinamide (29). Yield: 17%. H NMR (CDCl3) δ 8.72 (d, J =
6.0 Hz, 2H), 7.60 (d, J = 6.0 Hz, 2H), 7.03−7.07 (m, 2H), 6.93−6.95
(m, 2H), 6.81 (s, 1H), 6.43 (s, 2H), 1.92 (s, 6H); 13C NMR (CDCl3)
δ 163.48, 158.93 (d, J = 240.8 Hz), 158.75, 157.30, 152.19 (d, J = 2.4
Hz), 150.44, 147.75, 138.91, 138.71, 128.46, 120.95, 120.87 (d, J = 8.2
Hz), 116.69, 116.35 (d, J = 23.2 Hz), 112.42, 20.45; ESI−MS: m/z
420.2 (M + H)+.
In Vivo Xenografts. MDA-MB-231 (1 × 107 cells in matrigel/
mouse) tumor cells were subcutaneously injected into the right flank
of 5−8 week old female BALB/c nude (nu/nu) mice (BioLASCO,
Taiwan). Tumor volume was measured with a digital caliper once
tumor was palpable (within 10 to 15 days after implantation). The
tumor-bearing animals were treated when the size of the tumor
reached an average volume of ∼100 mm3. Fifteen mice were divided
into three groups and treated IV with compound 32 (20 mg/kg) or
vehicle control (5% DMSO and 10% Cremophor in H2O) at one dose
per day or PO with 32 (150 mg/kg) at two doses per day for 28 days.
Test compound 32 was formulated in a solution of 5% DMSO and
10% Cremophor in H2O. The size of tumor was measured by a digital
caliper and body weights of the mice were measured every 2 days. The
tumor size plotted in Figure 2 represents the average tumor volume
SE in cubic millimeters from five mice.
N-(4-{2,6-Dimethyl-4-[4-(trifluoromethyl)phenoxy]phenyl}-
thiazol-2-yl)isonicotinamide (30). Yield: 87%. 1H NMR (CDCl3) δ
8.76 (d, J = 6.0 Hz, 2H), 7.64 (d, J = 6.0 Hz, 2H), 7.59 (d, J = 8.6 Hz,
2H), 7.02 (d, J = 8.6 Hz, 2H), 6.86 (s, 1H), 6.57 (s, 2H), 1.98 (s, 6H);
13C NMR (CDCl3) δ 163.47, 159.87, 158.88, 155.34, 150.47, 147.55,
139.24, 138.61, 129.79, 127.12 (q, J = 3.8 Hz), 125.06 (q, J = 32.6 Hz),
124.11 (q, J = 271.5 Hz), 120.96, 118.35, 118.16, 112.57, 20.43; ESI−
MS: m/z 469.7 (M + H)+.
N-{4-[4-(4-Methoxyphenoxy)-2,6-dimethylphenyl]thiazol-2-
yl}isonicotinamide (31). Yield: 95%. 1H NMR (CDCl3) δ 8.71 (d, J
= 6.0 Hz, 2H), 7.60 (d, J = 6.0 Hz, 2H), 6.90−6.96 (m, 4H), 6.79 (s,
1H), 6.40 (s, 2H), 3.83 (s, 3H), 1.90 (s, 6H); 13C NMR (CDCl3) δ
163.81, 159.17, 158.09, 156.06, 150.15, 149.26, 147.59, 138.91, 138.63,
127.56, 121.15, 121.12, 115.96, 114.90, 112.32, 55.63, 20.44; ESI−MS:
m/z 431.7 (M + H)+.
Coimmunoprecipitation Analysis. K562 cells (2.5 × 106) were
seeded in a 100 mm dish under serum-free conditions. After 24 h, the
culture medium was changed to DMEM medium containing 10% FBS.
At the same time, cells were treated with DMSO, 32, 32 with MG132,
or MG132 for 16 h. At harvest, the medium was removed, and cells
were washed with ice-cold PBS. Cells were resuspended in ice-cold
Lysis 250 buffer (50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 5 mM
EDTA, 0.1% Triton X-100, 10 mM NaF, 1 mM phenylmethylsulfonyl
fluoride, and protease inhibitor cocktail (Sigma, P8340)) on ice for 30
min and then centrifuged at 12 000 rpm for 20 min at 4 °C. The
supernatants were collected for protein quantification and used for
coimmunoprecipitation. Briefly, 1.3 mg of total protein from each
sample was incubated with rabbit polyclonal anti-Nek2 antibody (5
μL, Rockland) or rabbit polyclonal anti-GFP antibody (5 μL, Santa
cruz) as IgG control at 4 °C. After incubation for 3 h, 100 μL of
Protein A Sepharose 4 Fast Flow (50% slurry, GE healthcare) was
added, and incubation continued overnight. The beads were collected,
washed three times with lysis buffer, and then boiled in a SDS-loading
buffer followed by SDS-PAGE. The presence of Hec1 and Nek2 were
detected by western blotting with mouse monoclonal antibodies
against Hec1 (GeneTex, Inc.) and Nek2 (BD Pharmingen).
Immunoblot Analysis. Cell lysates were prepared in 1× sample
buffer (SDS sample buffer containing 62.5 mM, pH 6.8, Tris-HCl, 2%
w/v SDS, 10% glycerol, 50 mM DTT, and 0.01% w/v bromophenol
blue). Samples were separated by SDS-PAGE, and proteins were
transferred onto PVDF membranes (GE Life Science, Piscataway, NJ,
USA). The membranes were then incubated with primary antibodies
in 3% bovine serum albumin in Tris-buffered saline with 0.1% Tween
20 (BSA-TBST). After washing in TBST, horseradish peroxidase-
conjugated secondary antibodies were used as a tracer, and the protein
signal was detected by enhanced chemiluminescence (Millipore,
Billerca, MA, USA). The following primary antibodies were used for
western blotting: cleaved caspase-3 (rabbit polyclonal, Cell Signaling
Technology), PARP (rabbit polyclonal, Cell Signaling Technology),
Mcl-1 (mouse monoclonal, BD Pharmingen), XIAP (rabbit polyclonal,
Cell Signaling Technology), Bcl-2 (mouse monoclonal, Santa Cruz
2-Fluoro-N-{4-[4-(4-methoxyphenoxy)-2,6-dimethylphenyl]-
1
thiazol-2-yl}isonicotinamide (32). Yield: 70%. H NMR (CDCl3,
400 MHz) δ 12.51 (brs, 1H), 8.28 (d, J = 5.2 Hz, 1H), 7.44 (d, J = 5.1
Hz, 1H), 7.22 (s, 1H), 6.96 (d, J = 9.2 Hz, 2H), 6.91 (d, J = 9.2 Hz,
2H), 6.79 (s, 1H), 6.37 (s, 2H), 3.82 (s, 3H), 1.86 (s, 6H); 13C NMR
(CDCl3) δ 162.02 (d, J = 239.2 Hz), 162.45 (d, J = 3.8 Hz), 159.07,
158.36, 156.14, 149.16, 148.35 (d, J = 14.6 Hz), 147.55, 144.22 (d, J =
7.5 Hz), 138.68, 127.18, 121.26, 118.87 (d, J = 4.6 Hz), 115.85,
114.92, 112.55, 108.04 (d, J = 39.1 Hz), 55.64, 20.35; ESI−MS: m/z
450.0 (M + H)+; HRMS calcd for C24H21FN3O3S (M+ + H),
450.1283; found, 450.1282.
N-{4-[4-(3-Methoxyphenoxy)-2,6-dimethylphenyl]thiazol-2-
yl}isonicotinamide (33). Yield: 51%. 1H NMR (CDCl3) δ 8.74 (d, J
= 6.0 Hz, 2H), 7.62 (d, J = 6.0 Hz, 2H), 7.23−7.26 (m, 1H), 6.82 (s,
1H), 6.67−6.69 (m, 1H), 6.51−6.56 (m, 2H), 6.47 (s, 2H), 3.81 (s,
3H), 1.94 (s, 6H); 13C NMR (CDCl3) δ 163.71, 160.93, 158.96,
157.69, 156.65, 150.41, 147.71, 138.73, 130.15, 128.47, 120.98, 117.37,
116.70, 112.41, 111.38, 108.95, 105.45, 55.40, 20.43; ESI−MS: m/z
431.6 (M + H)+.
N-{4-[4-(Benzo[d][1,3]dioxol-5-yloxy)-2,6-dimethylphenyl]-
thiazol-2-yl}isonicotinamide (34). Yield: 92%. 1H NMR (CDCl3) δ
8.73 (d, J = 5.8 Hz, 2H), 7.62 (d, J = 5.8 Hz, 2H), 6.81 (s, 1H), 6.78
(d, J = 8.5 Hz, 1H), 6.43−6.52 (m, 4H), 6.00 (s, 2H), 1.93 (s, 6H);
13C NMR (CDCl3) δ 163.38, 158.45, 157.80, 150.79, 150.57, 148.35,
147.89, 143.91, 138.82, 138.73, 128.14, 120.88, 116.39, 112.36, 112.23,
108.28, 102.32, 101.54, 20.47; ESI−MS: m/z 445.9 (M + H)+.
Cell Proliferation Assay. The antiproliferative activity of the
compounds with respect to HeLa, K562, MDA-MB-468, and MDA-
MB-231 was measured using the CellTiter96 assay kit (Promega)
following the manufacturer’s instruction. In brief, the cells were
maintained in normal glucose (1 μg/L) DMEM (Sigma, D5523)
containing 10% FBS and incubated at 37 °C in a 5% CO2 atmosphere.
Cells were plated at a density of 2500 cells/well in a 96-well plate for
24 h, treated with different concentrations of the compounds, and
incubated for another 96 h. At the end of the incubation, CellTiter 96
Aqueous One Solution Reagent (Promega) was added, and plates were
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Biotechnology, Inc.), Nek2 (mouse monoclonal, BD Pharmingen),
Hec1 (rabbit polyclonal, GeneTex, Inc.), and actin (mouse
monoclonal, Millipore).
Bassam, J.; Harrison, S. C. The Ndc80/HEC1 complex is a contact
point for kinetochore−microtubule attachment. Nat. Struct. Mol. Biol.
2007, 14, 54−59. (c) DeLuca, J. G.; Gall, W. E.; Ciferri, C.; Cimini, D.;
Musacchio, A.; Salmon, E. D. Kinetochore microtubule dynamics and
attachment stability are regulated by Hec1. Cell 2006, 127, 969−982.
(d) DeLuca, J. G.; Dong, Y.; Hergert, P.; Strauss, J.; Hickey, J. M.;
Salmon, E. D.; McEwen, B. F. Hec1 and Nuf2 are core components of
the kinetochore outer plate essential for organizing microtubule
attachment sites. Mol. Biol. Cell 2005, 16, 519−531. (e) Wei, R. R.;
Sorger, P. K.; Harrison, S. C. Molecular organization of the Ndc80
complex, an essential kinetochore component. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 5363−5367. (f) McCleland, M. L.; Gardner, R. D.;
Kallio, M. J.; Daum, J. R.; Gorbsky, G. J.; Burke, D. J.; Stukenberg, P.
T. The highly conserved Ndc80 complex is required for kinetochore
assembly, chromosome congression, and spindle checkpoint activity.
Genes Dev. 2003, 17, 101−114. (g) Hori, T.; Haraguchi, T.; Hiraoka,
Y.; Kimura, H.; Fukagawa, T. Dynamic behavior of Nuf2−Hec1
complex that localizes to the centrosome and centromere and is
essential for mitotic progression in vertebrate cells. J. Cell Sci. 2003,
116, 3347−3362. (h) Martin-Lluesma, S.; Stucke, V. M.; Nigg, E. A.
Role of Hec1 in spindle checkpoint signaling and kinetochore
recruitment of Mad1/Mad2. Science 2002, 297, 2267−2270.
(2) Cheeseman, I. M.; Chappie, J. S.; Wilson-Kubalek, E. M.; Desai,
A. The conserved KMN network constitutes the core microtubule-
binding site of the kinetochore. Cell 2006, 127, 983−997.
(3) Ciferri, C.; Musacchio, A.; Petrovic, A. The Ndc80 complex: Hub
of kinetochore activity. FEBS Lett. 2007, 581, 2862−2869.
(4) Foley, E. A.; Kapoor, T. M. Microtubule attachment and spindle
assembly checkpoint signalling at the kinetochore. Nat. Rev. Mol. Cell
Biol. 2013, 14, 25−37.
(5) Guimaraes, G. J.; Dong, Y.; McEwen, B. F.; Deluca, J. G.
Kinetochore-microtubule attachment relies on the disordered N-
terminal tail domain of Hec1. Curr. Biol. 2008, 18, 1778−1784.
(6) Miller, S. A.; Johnson, M. L.; Stukenberg, P. T. Kinetochore
attachments require an interaction between unstructured tails on
microtubules and Ndc80 (Hec1). Curr. Biol. 2008, 18, 1785−1791.
(7) Diaz-Rodriguez, E.; Sotillo, R.; Schvartzman, J.-M.; Benezra, R.
Hec1 overexpression hyperactivates the mitotic checkpoint and
induces tumor formation in vivo. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 16719−16724.
Immunofluorescent Staining, Microscopy, and Quantifica-
tion of Mitotic Abnormalities.28 For immunofluorescent staining,
cells were grown on Lab-Tek II Chamber Slide and washed with PBS
buffer (pH 7.4) before fixation with 4% paraformaldehyde. Following
permeabilization with 0.3% Triton X-100, cells were blocked with 5%
BSA/PBST and incubated with anti-α-tubulin antibodies. Then, DAPI
(4′,6′-diamidino-2-phenylindole) staining was applied, and cells were
mounted with ProLong Gold antifade (Invitrogen). Images were
examined with a NIKON 80i microscope at 400× or 1000×
magnification and captured with a Spot Digital Camera and Spot
Advanced Software Package (Diagnostic Instruments). The percentage
of cells with mitotic abnormalities was calculated by counting the
number of the cells showing the abnormal mitotic figures (including
chromosomal misalignment and formation of multipolar spindles)
over the total number of mitotic cells counted. A minimum of 500 cells
from randomly selected fields were scored per condition per
experiment.
ASSOCIATED CONTENT
■
S
* Supporting Information
Protocols for the kinase, [3H]astemizole competitive binding,
solubility, and metabolic stability assays for compound 32. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +886 5 271 7959. Fax: +886 5 271 7901. E-mail:
lukehuang@mail.ncyu.edu.tw and lukehuang0226@gmail.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Wen-Hwa Lee, Phang-Lang Chen, and Yumay
Chen for their encouragement to initiate the project. We thank
Drs. Chia-Lin Jeff Wang, Meng-Hsin Chen, Henry Feng,
Horace H. Loh, Jui-Lien Huang, and Chi-feng Chang and Mrs.
Lihyan Lee for their great leadership and support on the
project. We are grateful to the Ministry of Economic Affairs of
the Republic of China, Taivex Therapeutics Corporation, and
the Ministry of Science and Technology of the Republic of
China (102-2113-M-415-005-MY2) for financial support.
(8) Chen, Y.; Riley, D. J.; Chen, P.-L.; Lee, W.-H. HEC, a novel
nuclear protein rich in leucine heptad repeats specifically involved in
mitosis. Mol. Cell. Biol. 1997, 17, 6049−6056.
(9) van’t Veer, L. J.; Dai, H. Y.; van de Vijver, M. J.; He, Y. D. D.;
Hart, A. A. M.; Mao, M.; Peterse, H. L.; van der Kooy, K.; Marton, M.
J.; Witteveen, A. T.; Schreiber, G. J.; Kerkhoven, R. M.; Roberts, C.;
Linsley, P. S.; Bernards, R.; Friend, S. H. Gene expression profiling
predicts clinical outcome of breast cancer. Nature 2002, 415, 530−536.
(10) Glinsky, G. V.; Berezovska, O.; Glinskii, A. B. Microarray
analysis identifies a death-from-cancer signature predicting therapy
failure in patients with multiple types of cancer. J. Clin. Invest. 2005,
115, 1503−1521.
(11) Mo, Q.-q; Chen, P.-b; Jin, X.; Chen, Q.; Tang, L.; Wang, B.-b.;
Li, K.-z.; Wu, P.; Fang, Y.; Wang, S.-x.; Zhou, J.-f.; Ma, D.; Chen, G.
Inhibition of Hec1 expression enhances the sensitivity of human
ovarian cancer cells to paclitaxel. Acta Pharmacol. Sin. 2013, 34, 541−
548.
(12) Gurzov, E. N.; Izquierdo, M. RNA interference against Hec1
inhibits tumor growth in vivo. Gene Ther. 2006, 13, 1−7.
(13) Wei, R.; Ngo, B.; Wu, G.; Lee, W.-H. Phosphorylation of the
Ndc80 complex protein, HEC1, by Nek2 kinase modulates
chromosome alignment and signaling of the spindle assembly
checkpoint. Mol. Biol. Cell 2011, 22, 3584−3594.
DEDICATION
In memory of Dr. Paonien Chen.
■
ABBREVIATIONS USED
■
Co-IP, coimmunoprecipitation; Hec1, highly expressed in
cancer 1; Nek2, never in mitosis gene a (NIMA)-related kinase
2; t-BuXPhos, 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbi-
phenyl; IV, intravenous; PBS, phosphate buffered saline; PO,
per os; TBABr3, tetrabutylammonium tribromide; hERG,
human ether-a-go-go-related gene; CML, chronic myelogenous
leukemia
REFERENCES
■
(14) Chen, Y.; Riley, D. J.; Zheng, L.; Chen, P.-L.; Lee, W.-H.
Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase
is essential for faithful chromosome segregation. J. Biol. Chem. 2002,
277, 49408−49416.
(15) Du, J.; Cai, X.; Yao, J.; Ding, X.; Wu, Q.; Pei, S.; Jiang, K.;
Zhang, Y.; Wang, W.; Shi, Y.; Lai, Y.; Shen, J.; Teng, M.; Huang, H.;
(1) For the biological functions of Hec1, see (a) Varma, D.;
Chandrasekaran, S.; Sundin, L. J. R.; Reidy, K. T.; Wan, X.; Chasse, D.
A. D.; Nevis, K. R.; DeLuca, J. G.; Salmon, E. D.; Cook, J. G.
Recruitment of the human Cdt1 replication licensing protein by the
loop domain of Hec1 is required for stable kinetochore−microtubule
attachment. Nat. Cell Biol. 2012, 14, 593−604. (b) Wei, R. R.; Al-
4109
dx.doi.org/10.1021/jm401990s | J. Med. Chem. 2014, 57, 4098−4110

Journal of Medicinal Chemistry
Article
Fei, Q.; Reddy, E. S.; Zhu, J.; Jin, C.; Yao, X. The mitotic checkpoint
kinase NEK2A regulates kinetochore microtubule attachment stability.
Oncogene 2008, 27, 4107−4114.
(16) Qiu, X.-L.; Li, G.; Wu, G.; Zhu, J.; Zhou, L.; Chen, P.-L.;
Chamberlin, A. R.; Lee, W.-H. Synthesis and biological evaluation of a
series of novel inhibitor of Nek2/Hec1 analogues. J. Med. Chem. 2009,
52, 1757−1767.
(17) Wu, G.; Qiu, X.-L.; Zhou, L.; Zhu, J.; Chamberlin, A. R.; Lau, J.;
Chen, P.-L.; Lee, W.-H. Small molecule targeting the Hec1/Nek2
mitotic pathway suppresses tumor cell growth in culture and in animal.
Cancer Res. 2008, 68, 8393−8399.
́
(18) Repichet, S.; Le Roux, C.; Roques, N.; Dubac, J. BiCl3-catalyzed
Friedel−Crafts acylation reactions: bismuth(III) oxychloride as a water
insensitive and recyclable procatalyst. Tetrahedron Lett. 2003, 44,
2037−2040.
(19) Lee, L. F.; Schleppnik, F. M.; Howe, R. K. Syntheses and
reactions of 2-halo-5-thiazolecarboxylates. J. Heterocycl. Chem. 1985,
22, 1621−1630.
(20) Anderson, K. W.; Ikawa, T.; Tundel, R. E.; Buchwald, S. L. The
selective reaction of aryl halides with KOH: synthesis of phenols,
aromatic ethers, and benzofurans. J. Am. Chem. Soc. 2006, 128, 10694−
10695.
(21) Hames, R. S.; Wattam, S. L.; Yamano, H.; Bacchieri, R.; Fry, A.
M. APC/C-mediated destruction of the centrosomal kinase Nek2A
occurs in early mitosis and depends upon a cyclin A-type D-box.
EMBO J. 2001, 20, 7117−7127.
(22) Hames, R. S.; Crookes, R. E.; Straatman, K. R.; Merdes, A.;
Hayes, M. J.; Faragher, A. J.; Fry, A. M. Dynamic recruitment of Nek2
kinase to the centrosome involves microtubules, PCM-1, and localized
proteasomal degradation. Mol. Biol. Cell 2005, 16, 1711−1724.
(23) The radiometric assays were conducted in similar manner to
that of our previously published Aurora kinase assay, see: Chiang, C.-
C.; Lin, Y.-H.; Lin, S. F.; Lai, C.-L.; Liu, C.; Wei, W.-Y.; Yang, S.-c.;
Wang, R.-W.; Teng, L.-W.; Chuang, S. H.; Chang, J.-M.; Yuan, T.-T.;
Lee, Y.-S.; Chen, P.; Chi, W.-K.; Yang, J.-Y.; Huang, H.-J.; Liao, C.-B.;
Huang, J.-J. Discovery of pyrrole-indoline-2-ones as Aurora kinase
inhibitors with a different inhibition profile. J. Med. Chem. 2010, 53,
5929−5941.
(24) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small
molecule kinase inhibitors. Nat. Rev. Cancer 2009, 9, 28−39.
(25) Finlayson, K.; Turnbull, L.; January, C. T.; Sjarkey, J.; Kelly, J. S.
[3H]Dofetilide binding to HERG transfected membranes: a potential
high throughput preclinical screen. Eur. J. Pharmacol. 2001, 430, 147−
148.
(26) Chiu, P. J. S.; Marcoe, K. F.; Bounds, S. E.; Lin, C.-H.; Feng, J.-
J.; Lin, A.; Cheng, F.-C.; Crumb, W. J.; Mitchell, R. Validation of a
[3H]astemizole binding assay in HEK293 cells expressing HERG K+
channels. J. Pharmacol. Sci. 2004, 95, 311−319.
(27) Yao, X.; Anderson, D. L.; Ross, S. A.; Lang, D. G.; Desai, B. Z.;
Cooper, D. C.; Wheelan, P.; McIntyre, M. S.; Bergquist, M. L.;
MacKenzie, K. I.; Becherer, J. D.; Hashim, M. A. Predicting QT
prolongation in humans during early drug development using hERG
inhibition and an anaesthetized guinea-pig model. Br. J. Pharmacol.
2008, 154, 1446−1456.
(28) Wu, G.; Wei, R.; Cheng, E.; Ngo, B.; Lee, W.-H. Hec1
contributes to mitotic centrosomal microtubule growth for proper
spindle assembly through interaction with Hice1. Mol. Biol. Cell 2009,
20, 4686−4695.
4110
dx.doi.org/10.1021/jm401990s | J. Med. Chem. 2014, 57, 4098−4110