Modification and biological evaluation of thiazole derivatives as novel inhibitors of metastatic cancer cell migration and invasion

Article abstract of DOI:10.1021/jm500724x

Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent anti

Full text of DOI:10.1021/jm500724x

Article
pubs.acs.org/jmc
Open Access on 07/09/2015
Modification and Biological Evaluation of Thiazole Derivatives as
Novel Inhibitors of Metastatic Cancer Cell Migration and Invasion
Shilong Zheng,*,† Qiu Zhong,‡ Yulan Xi,∥ Madhusoodanan Mottamal,† Qiang Zhang,†
Richard L. Schroeder,‡ Jayalakshmi Sridhar,‡ Ling He,∥ Harris McFerrin,§ and Guangdi Wang*,†,‡
‡
§
†RCMI Cancer Research Center, Department of Chemistry, and Department of Biology, Xavier University of Louisiana, New
Orleans, Louisiana 70125, United States
∥West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 600041, China
ABSTRACT: Fascin has recently emerged as a potential therapeutic target, as its expression in cancer cells is closely associated
with tumor progression and metastasis. Following the initial discovery of a series of thiazole derivatives that demonstrated potent
antimigration and antiinvasion activities via possible inhibition of fascin function, we report here the design and synthesis of 63
new thiazole derivatives by further structural modifications in search of more potent fascin inhibitors. The 5 series of analogues
with longer alkyl chain substitutions on the thiazole nitrogen exhibited greater antimigration activities than those with other
structural motifs. The most potent analogue, 5p, inhibited 50% of cell migration at 24 nM. Moreover, the thiazole analogues
showed strong antiangiogenesis activity, blocking new blood vessel formation in a chicken embryo membrane assay. Finally, a
functional study was conducted to investigate the mechanism of action via interaction with the F-actin bundling protein fascin.
optimize the structure−activity relationships, we have designed
and synthesized 63 additional thiazole derivatives where we
sought to (1) homologize the two lead structures by varying the
substitution on the thiazole nitrogen, (2) change the linker
structure between thiazole and phenyl groups, and (3) modify
other substitution groups on both the thiazole ring and the
phenyl rings (Figure 1). These thiazole analogues were then
biologically evaluated to determine their cytotoxicity, anti-
migration, and antiinvasion activities. Further, molecular
modeling was performed to assist in the elucidation of observed
structure−activity-relationships. For the most potent thiazole
derivatives, an in vivo assay utilizing chick embryo chorioallan-
toic membrane (CAM) was performed to assess their in vivo
antimetastasis potential by inhibiting angiogenesis. Finally we
investigated their mode of action by overexpressing the protein
fascin in cancer cell lines to determine if the activities of the
compounds can abrogate the enhanced migration and invasion
of the transfected cell lines.
INTRODUCTION
■
The actin-bundling protein fascin has been linked to tumor
progression, invasion, and metastasis, a fatal development of the
disease.1−5 Fascin has recently emerged as a novel therapeutic
target for treatment of cancer metastasis.6−8 A cell-based fascin
bioassay identified compounds with potential antimetastasis
functions, with diverse chemical structural features.9 However,
no follow-up studies of biological activities were reported to
confirm that the compounds indeed target fascin to inhibit
migration, invasion, and metastasis.9
From shape-based molecular modeling and subsequent
rational design and synthesis, we have recently identified a
thiazole lead compound 1 (Figure 1) that effectively blocked
cell migration and invasion via interactions with the protein
fascin.10 With potent activities (IC50 in the 100 nM range) in
inhibiting migration and invasion of several metastatic human
breast cancer cell lines such as MDA-MB-231, HeLa, and A549,
this compound exhibited no cytotoxicity at concentrations
exceeding 100 μM. The finding of thiazole compounds as
antimigration and antiinvasion agents opened up new
possibilities of fascin-targeting molecules that can be further
optimized for greater potency and bioavailability while
maintaining minimal cytotoxicity. To further explore and
Received: May 9, 2014
Published: July 9, 2014
© 2014 American Chemical Society
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Figure 1. Structures of lead compounds 1 and 2 and their analogues from lead modifications.
Scheme 1. Synthesis of Analogues of Lead Compounds
the literature procedure.10 4-(2,4-Dimethylphenyl)thiazol-2-
amine (9) was treated with benzenesulfonic chloride to give
10 which was transformed to the analogue 11 and its isomer 12
by the N-methylation reaction in THF. The acylation of the 2-
RESULTS AND DISCUSSION
■
Chemistry. As shown in Scheme 1, compounds 5 and 7 and
their isomers 6 and 8 were respectively obtained from the N-
alkylation of the amides 3 and 4 which were prepared following
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Table 1. Antimigration Efficacy and Cytotoxicity of the Synthetic Thiazole Derivatives
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Table 1. continued
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Table 1. continued
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Table 1. continued
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Table 1. continued
aminothiazoles 13 by acyl chloride provided the amides 14 at
room temperature in dichloromethane, and further methylation
of 14 led to the desired analogues 15 and their corresponding
isomers 16.
values going from a low of 24 nM to greater than 50 μM or no
apparent activity. Such variations in activity appears to be
dependent on minor structural modifications, revealing some
interesting trends that may help our understanding of the
structure−activity-relationships for further optimization of
pharmacological index of thiazole derivatives.
Compounds 5l−r are homologues from the lead structure 1
that were designed and prepared to investigate the effect of
thiazole-N substitution on gain or loss of the antimigration
activity. For comparison, 1, the most potent thiazole derivative
from our previous study,10 was also included in the assay. When
the thiazole N-methyl is changed to an ethyl group (5l), there
was a marked decrease of activity from 0.218 to 1.945 μM.
Interestingly, an n-propyl substitution at the same position
(5m) confers similar antimigration activity with an IC50 of
0.292 μM. This trend continues as the alkyl chain length
Antimigration Activity and Cytotoxicity. A Transwell
migration assay was used to determine the effects of the
synthesized thiazole derivatives on the migratory capacity of
MDA-MB-231, an invasive and metastatic breast cancer cell
line. The cancer cells were seeded at a density of 2.5 × 104 per
well in the upper chamber in serum-free media. In the presence
of varying concentrations of the thiazole derivatives, the cells’
ability to migrate to the lower chamber with media containing
5% FBS was measured by counting the total number of cells in
the lower chamber after 24 h. The concentration of individual
thiazole compounds at which inhibition of migration is
observed at 50% is defined as the IC50 value listed in Table
1. To evaluate possible contributions of cell viability loss to
reduced migration, the thiazole derivatives were also subjected
to cell survival assays to determine their cytotoxicities. These
were conducted by treating the MDA-MB-231 cells with
individual thiazole compounds for 5 days and counting the
surviving cells.
increased to n-butyl (5n, IC50 = 0.196 μM), n-hexyl (5o, IC50
=
0.045 μM), and n-dodecyl (5p, IC50 = 0.024 μM). In addition,
allyl substitution at the thiazole nitrogen (5q) yielded activity
comparable to n-propyl (5m), but changing the substitution
(5r) to propynyl leads to a significant loss of the activity,
resulting in an IC50 value of 3.89 μM. On another note, putting
the propynyl group on the amide nitrogen (6) appears to
restore the activity.
The 63 newly synthesized thiazole derivatives displayed a
wide range of antimigration activities as reflected in the IC50
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Figure 2. Antiinvasion activities of selected thiazole compounds.
Attempt to modify the amide linker leads to mixed results. If
the amide linker is replaced by a sulfamide structure,
antimigration activity was largely lost in 12 but not in 11.
The single structural difference between 11 and 12 is the
methyl substitution. Thiazole N-methyl substitution confers,
whereas the amide N-methyl substitution deprives, antimigra-
tion activity.
The lead compound 2 was a potent thiazole derivative
discovered in a previous study, which also displayed significant
cytotoxicity in several cancer cell lines at 10 μM.10 In the
current study we further investigated if modifications on N-alkyl
substitutions could improve the activity and toxicity profile.
Thus, when the alkyl substitution at the thiazole nitrogen varied
from methyl (5j), ethyl (7a), propyl (7b) to allyl (7c) and
propynyl (7d), the cytotoxicity of the compounds remained
significant, with relative survival ratio after 5-day treatment at
10 μM ranging from 20.4% to 70.9%. However, the cytotoxicity
is largely abrogated when the methyl and ethyl substitution is
on the amide nitrogen (4e, 8a, 8b) while strong antimigration
activity was retained. It was also noted that these compounds
(7a−d) exhibited moderate to strong inhibition of cell growth,
suggesting that, in addition to blocking fascin-mediated
migration, these compounds may have other molecular targets
that regulate cell survival and growth.
Next we examine how modifications on the thiazole ring
affect antimigration activity. A methyl substitution on the 5-
position of the thiazole ring (14h) results in a near complete
loss of activity. Adding a fluorine atom at the para-position of
the 1-thiazole phenyl brings back cytotoxicity without a
significant gain of antimigration activity (14i). An attempt to
move the thiazole phenyl ring to the 5-position results in a
more linear analogue that has stronger growth inhibitory effect
but also more antimigratory (14j). Interestingly, adding a
thiazole N-methyl group to the above three analogues gives a
more potent 15h (compared to 14h) and 15i (compared to
15i) but an inactive 15j (compared to 14j).
In comparison of 15j (thiazole N-methyl) to 16f (amide N-
methyl), it was noted that the analogue with amide N-methyl
substitution displayed significantly greater toxicity and anti-
migration activity than that with a thiazole N-methyl group.
Modifications on the thiazole phenyl ring by halogen and
trifluoromethyl substitutions afforded six analogues 14b, 14c,
14d, 14e, 14f, 14g, of which only 14e and 14f showed strong
antimigration activities with moderate inhibition of cell growth
at the same time. It was noted that chlorine substitution at meta
position (14e) is far superior to ortho position (14b). Adding a
methyl group on the thiazole nitrogen to some of these
analogues appears to endow more potent cytotoxicity while
increasing the overall inhibitory effect on cell motility. Thus,
15b. 15c, 15d, 15e, 15f, and 15g all displayed potent
antimigration activities with IC50 values ranging from 0.096
μM (15f) to 1.07 μM (15g).
Attempts to modify both thiazole phenyl and amide phenyl
ring yielded eight analogues (14k through 14r). These
compounds showed almost no antimigration activities except
14o with an IC50 value of 1.03 μM. However, if these analogues
are modified further by adding a thiazole N-methyl substitution
(15k through 15m), some modest increases in the activity are
obtained. For example, 15l exhibited an IC50 value of 0.758 μM.
On the other hand, if the methyl substitution is on the amide
nitrogen (16g through 16m), the results are mixed. For
instance, compared to 14k, IC50 value of 16g improved from
>25 to 0.312 μM, an increase of potency by nearly 100-fold.
Also, from 15o to 16j, the position change of methyl
substitution from thiazole nitrogen to amide nitrogen resulted
in an increase of antimigration activity by 4 times.
Antiinvasion. To determine the antiinvasion activities of
the thiazole compounds, the most potent antimigration
analogues were selected for a Matrigel invasion assay. As
shown in Figure 2, at 10 μM, all tested compounds
demonstrate 50% or greater inhibition of cell invasion through
the Matrigel. Notably, compound 5o exhibited the greatest
potency in blocking the cancer cell invasion. It is also noted
that the antiinvasion activities of the selected thiazole
derivatives do not always correlate with their antimigration
activities.
Antiangiogenesis. Given the association of cell migration
with angiogenesis11−14 and the possible involvement of fascin
in angiogenesis,15 we next investigated if the thiazole
compounds might have any antiangiogenic properties using
the chick chorioallantoic membrane (CAM) assay. In this test
the vascular system of a fertilized chicken embryo is used as a
model. Figure 3 demonstrates the effects of 5o and 5p on the
development of embryonal blood vessels compared to a
negative control (PBS) and a positive control (10 ng/plug
basic fibroblast growth factor (bFGF) and 30 ng/plug vascular
endothelial growth factor (VEGF)) without the thiazole
compounds. The antiangiogenesis activity of the three thiazole
compounds was determined by the suppression of angiogenic
action of BV (bFGF + VEGF) when each compound was
added to a collagen containing BV and placed on the
chorioallantoic membrane of 10-day old chick embryos for 3
days. As shown in Figure 3, the analogues 5o and 5p were seen
to potently block the angiogenic action of BV.
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Figure 5. Enhanced cell migration induced by fascin-overexpression is
abrogated by thiazole analogues 5l, 5o, and 5p.
Figure 3. Effect of 5o and 5p on angiogenesis in chick embryo ex ovo
chorioallantoic membrane (CAM) assay. Angiogenesis was scored by
two scorers in a blinded fashion on a scale from 0 to 3 where 0
contained no new vessels and 3 exhibited extreme angiogenesis
determined by the presence of new vessels radiating out from the plug
in addition to the regular repeating pattern of the normal CAM
vasculature. Dashed lines in representative images (below) indicate the
top edges of the collagen and drug-containing plugs from which
angiogenesis was determined. In these experiments, the thiazole
analogues 5o and 5p significantly inhibited the induction of
angiogenesis by bFGF and VEGF (BV) after 3 days.
about the same percentage (Figure 5). These results provided
additional evidence that the thiazole derivatives likely acted
through interaction with fascin.
Effect on F-Actin Structure. To investigate if the thiazole
analogues inhibit cell migration and invasion by impairing the
F-actin network, actin filaments of HeLa cells treated with
selected thiazole analogues were stained with phalloidin tagged
with fluorescent agent (Acti-stain 488 fluorescent phalloidin).
As shown in Figure 6, the control cells showed a distinct
presence of F-actin filaments along the cells and had more
evenly distributed F-actin structures in the cytoplasm. In
contrast, after treatment with the three thiazole analogues 5o,
5p, and 8b each at 10−6 M, the cells exhibited various degrees
of disorganization of actin filaments and a decrease of F-actin
staining intensity near the cell membranes. These results
suggest that the antimigration and antiinvasion activities of the
thiazole analogues may be mediated by interfering with the
actin filament network.
Mechanism of Action. Our previous study10 found that
the thiazole derivatives blocked migration and invasion of
cancer cells by impairing the cytoskeleton dynamics accom-
panied by reduced colocalization of the actin-bundling protein
fascin. To provide further evidence that fascin might be
involved in the antimigratory action of the newly synthesized
thiazole analogues, we overexpressed fascin in MDA-MB-231
cells to investigate the resulting cell motility and the effect of
the thiazole compounds on the cells overexpressing fascin.
Figure 4 shows the higher level of fascin expression in the
transfected cells than the native level of fascin in the control
cells (Figure 4A). Consistent with previous findings,3,16−23 the
fascin+ cells exhibited significantly higher (2.4-fold increase)
migration than the control cells (vector). Treatment of the
fascin+ cells with our most potent thiazole analogues, 5o and
5p, nearly completely abrogated the enhanced migration as
illustrated in Figure 5. At 10 μM 5o and 5p, the control MDA-
MB-231 cells lost about 80% migratory capacity. The thiazole
compounds also inhibited the fascin+ MDA-MB-231 cells by
CONCLUSION
■
In summary, the 63 new thiazole derivatives designed and
prepared by further structural modifications of the previously
discovered lead compounds 1 and 2 produced more potent
fascin inhibitors. Among the active compounds, the 5 series of
analogues with longer alkyl chain substitutions on the thiazole
nitrogen exhibited greater antimigration activities than those
with other structural motifs. The most potent analogue, 5p,
inhibited 50% of cell migration at a concentration of 24 nM. As
expected, all thiazole derivatives that were selected for Matrigel
Figure 4. Overexpressing fascin in MDA-MB-231 leads to enhanced migration.
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Figure 6. Effects of thiazole analogues 5o, 5p, and 8b on the F-actin structure of HeLa cells. Cells were plated on glass coverslips and incubated with
vehicle (DMSO), 5o, 5p, or 8b for 24 h and were fixed and stained with Acti-stain 488 phalloidin. Cells were observed under a fluorescent
microscope, a digital CCD camera, and 40× objective. Data are representative of three independent experiments.
added to quench the reaction, and the mixture was further diluted with
water (50 mL). The mixture was extracted with CH2Cl2 (3 × 50 mL).
The combined organic phase was dried with anhydrous MgSO4. After
removal of all the solvent, the residue was purified by silica gel
chromatography (hexane/EtOAc = 4:1) to afford products 5 (more
polar) and 6 or 7 (more polar) and 8 as solids.
N-(4-(2,4-Dimethylphenyl)-3-ethylthiazol-2(3H)-ylidene)-
benzamide (5l). 1H NMR (300 MHz, CDCl3): 8.37 (dd, J = 1.8 and
8.1 Hz, 2H), 7.49−7.42 (m, 3H), 7.18−7.10 (m, 3H), 6.45 (s, 1H),
4.35 (m, 1H), 3.82 (m, 1H), 1.24 (t, J = 6.9 Hz, 3H). 13C NMR (75
MHz, CDCl3): 174.1, 167.8, 140.1, 137.82, 137.75, 137.2, 131.3, 131.2,
130.6, 129.2, 128.0, 127.2, 126.8, 106.8, 42.1, 21.3, 19.6, 13.8. GC−
MS: 336 (M+). HRMS (ESI(+)) calcd for C20H21N2OS (M + H):
337.1375. Found: 337.1365.
invasion assays were found to have potent antiinvasion
activities. In addition to antimigration and antiinvasion
properties, two representative thiazole derivatives, 5o and 5p,
also showed strong antiangiogenesis activity, blocking new
blood vessel formation in a chicken embryo membrane assay. A
functional study in which enhanced cell motility due to fascin
overexpression was nearly completely abrogated by the most
potent analogues 5o and 5p provided additional evidence that
the action of the thiazole derivatives involved targeting the F-
actin bundling protein fascin. This proposed mechanism of
action is further supported by the observation that cellular F-
actin structures were significantly impaired by the treatment of
the thiazole analogues.
N-(4-(2,4-Dimethylphenyl)-3-propylthiazol-2(3H)-ylidene)-
benzamide (5m). 1H NMR (300 MHz, CDCl3): 8.36 (m, 2H),
7.52−7.42 (m, 3H), 7.17−7.09 (m, 3H), 6.45 (s, 1H), 4.28 (m, 1H),
3.71 (m, 1H), 2.41 (s, 3H), 2.13 (s, 3H), 1.70 (m, 2H), 0.81 (t, J = 7.5
Hz, 3H). 13C NMR (75 MHz, CDCl3): 174.0, 168.1, 140.0, 138.2,
137.7, 137.2, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8, 106.8,
48.5, 21.8, 21.3, 19.6, 11.2. GC−MS: 350 (M+). HRMS (ESI(+)) calcd
for C21H23N2OS (M + H): 351.1531. Found: 351.1525.
N-(3-Butyl-4-(2,4-dimethylphenyl)thiazol-2(3H)-ylidene)-
benzamide (5n). 1H NMR (300 MHz, CDCl3): 8.36 (m, 2H), 7.52−
7.42 (m, 3H), 7.17−7.09 (m, 3H), 6.45 (s, 1H), 4.33 (m, 1H), 3.73
(m, 1H), 2.40 (s, 3H), 2.14 (s, 3H), 1.64 (m, 2H), 1.22 (m, 2H), 0.81
(t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): 174.1, 168.1, 140.1,
138.2, 137.7, 137.2, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8,
106.9, 46.7, 30.4, 21.3, 19.9, 19.6, 13.6. GC−MS: 364 (M+). HRMS
(ESI(+)) calcd for C22H25N2OS (M + H): 365.1688. Found:
365.1682.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were purchased from AK
Scientific, Sigma-Aldrich Chemical Co., Fisher Scientific, ACROS, and
Pharmco-AAPER and were used as received. All organic solvents
(Pharmco-AAPER) used were of reagent grade quality and were used
without further purification. NMR spectra were recorded on a Bruker
Fourier-300 spectrometer (Bruker Inc., Billerica, MA) in ppm. Crude
synthetic products were purified by the following methods:
chromatography on silica gel (60−100 mesh, Fisher Scientific)
column. Analytical thin layer chromatography (TLC) was performed
on 250 μm fluorescent plates (Agela Technologies, DE, USA) and
visualized by using UV light. For all products, the purity was
ascertained to be greater than 95% by the HPLC method using a
Shimadzu (Columbia, MD) 2010 HPLC−UV/MS system with a C-18
reverse phase column and by GC−MS analyses using an Agilent
Technologies 5975C inert MSD mass spectrometer.
N-(4-(2,4-Dimethylphenyl)-3-hexylthiazol-2(3H)-ylidene)-
benzamide (5o). 1H NMR (300 MHz, CDCl3): 8.36 (dd, J = 1.5 and
7.8 Hz, 2H), 7.52−7.42 (m, 3H), 7.17−7.09 (m, 3H), 6.45 (s, 1H),
4.33 (m, 1H), 3.72 (m, 1H), 2.41 (s, 3H), 2.14 (s, 3H), 1.61 (m, 2H),
1.23−1.17 (m, 6H), 0.81 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz,
CDCl3): 174.1, 168.0, 140.1, 138.1, 137.7, 137.2, 131.3, 131.2, 130.7,
129.2, 128.0, 127.3, 126.8, 106.9, 46.9, 31.1, 28.2, 26.2, 22.4, 21.3, 19.6,
General Procedure for N-Alkylation of 3 and 4. To a cooled
mixture of NaH (0.26 g, 60% in oil, 6.5 mmol) in THF (20 mL) was
added a solution of compound 3 or 4 (5 mmol) in THF (10 mL)
dropwise. The mixture was warmed to room temperature and stirred
for 20 min. After that, the mixture was cooled to 0 °C again and MeI
or RBr (6.5 mmol) was added dropwise. The mixture was then
warmed to room temperature and stirred for 2 h. Water (5 mL) was
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13.9. GC−MS: 392 (M+). HRMS (ESI(+)) calcd for C24H29N2OS (M
+ H): 393.2001. Found: 393.1986.
7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): 170.6, 159.0, 149.5, 135.2,
134.8, 130.4, 128.68, 128.65, 127.8, 126.7, 126.0, 109.2, 45.2, 14.1.
GC−MS: 308 (M+). HRMS (ESI(+)) calcd for C18H17N2OS (M +
H): 309.1062. Found: 309.1053.
N-(4-(2,4-Dimethylphenyl)-3-dodecylthiazol-2(3H)-ylidene)-
benzamide (5p). 1H NMR (300 MHz, CDCl3): 8.35 (m, 2H), 7.49−
7.26 (m, 3H), 7.17−7.09 (m, 3H), 6.44 (s, 1H), 4.32 (m, 1H), 3.73
(m, 1H), 2.40 (s, 3H), 2.13 (s, 3H), 1.59−1.50 (m, 4H), 1.28−1.17
(m, 16H), 0.88 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3):
174.1, 168.1, 140.0, 138.1, 137.7, 137.3, 131.3, 131.2, 130.7, 129.2,
128.0, 127.3, 126.8, 106.9, 46.9, 32.8, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3,
28.9, 28.2, 26.5, 25.8, 22.7, 21.3, 19.6, 14.1. MS (ESI): 477 (M + H).
HRMS (ESI(+)) calcd for C30H41N2OS (M + H): 477.2940. Found:
477.2945.
N-(3-Allyl-4-(2,4-dimethylphenyl)thiazol-2(3H)-ylidene)-
benzamide (5q). 1H NMR (300 MHz, CDCl3): 8.35 (m, 2H), 7.51−
7.41 (m, 3H), 7.14−7.07 (m, 3H), 6.46 (s, 1H), 5.87 (m, 1H), 5.10
(dd, J = 1.2 and 10.2 Hz, 1H), 4.98 (dd, J = 1.2 and 17.1 Hz, 2H), 4.42
(m, 1H), 2.40 (s, 3H), 2.13 (s, 3H). 13C NMR (75 MHz, CDCl3):
174.2, 168.1, 140.1, 137.8, 137.1, 131.3, 131.1, 130.8, 129.2, 128.0,
127.0, 126.6, 118.4, 106.8, 49.1, 21.3, 19.7. GC−MS: 348 (M+).
HRMS (ESI(+)) calcd for C21H21N2OS (M + H): 349.1375. Found:
349.1370.
N-(4-Phenylthiazol-2-yl)-N-propylbenzamide (8b). 1H NMR
(300 MHz, CDCl3): 7.91 (dd, J = 1.5 and 6.9 Hz, 2H), 7.53−7.32 (m,
8H), 7.24 (s, 1H), 4.18 (q, J = 7.5 Hz, 2H), 1.83 (m, 2H), 0.83 (t, J =
7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): 170.8, 159.3, 149.5, 135.2,
134.8, 130.4, 128.68, 128.66, 128.5, 127.9, 127.0, 126.0, 109.2, 51.6,
21.9, 11.2. GC−MS: 322 (M+). HRMS (ESI(+)) calcd for
C19H19N2OS (M + H): 323.1218. Found: 323.1211.
Synthesis of N-(4-(2,4-Dimethylphenyl)thiazol-2-yl)-
benzenesulfonamide (10). To a solution of 9 (0.61g, 3.0 mmol)
and pyridine (0.96 mL, 12 mmol) in anhydrous dichloromethane was
added benzenesulfonic chloride (0.76 mL, 6 mmol) in dichloro-
methane dropwise at 0 °C. After being stirred at room temperature for
2 h under nitrogen atmosphere, the reaction mixture was concentrated
in vacuo. The saturated Na2CO3 solution was added to quench the
reaction, and the solution was extracted with ethyl acetate, dried over
MgSO4, and concentrated in vacuo. The residue was purified by flash
1
chromatography to give product 10 (0.57 g, 55% yield) as a solid. H
N-(4-(2,4-Dimethylphenyl)-3-(prop-2-yn-1-yl)thiazol-2(3H)-
NMR (300 MHz, CDCl3): 9.85 (bs, 1H), 7.93 (d, J = 7.8 Hz, 2H),
7.53−7.43 (m, 3H), 7.16 (d, J = 7.8 Hz, 1H), 7.08−7.04 (m, 3H), 6.26
(s, 1H), 2.34 and 2.32 (ds, 6H). 13C NMR (75 MHz, CDCl3): 168.7,
141.9, 140.1, 136.3, 136.1, 132.2, 132.0, 128.9, 128.7, 127.2, 126.5,
125.7, 103.6, 21.2, 20.4. GC−MS: 344 (M+). HRMS (ESI(+)) calcd
for C17H17N2O2S2 (M + H): 345.0731. Found: 345.0727.
1
ylidene)benzamide (5r). H NMR (300 MHz, CDCl3): 8.40 (m,
2H), 7.52−7.42 (m, 3H), 7.23−7.11 (m, 3H), 6.47 (s, 1H), 4.80 (m,
2H), 2.40 (s, 3H), 2.21 (m, 1H), 2.19 (s, 3H). 13C NMR (75 MHz,
CDCl3): 174.4, 168.1, 140.4, 138.1, 137.1, 136.9, 131.6, 131.4, 130.8,
129.4, 128.1, 126.9, 126.4, 106.9, 77.3, 72.4, 36.0, 21.4, 19.9. MS (ESI):
347 (M + H). HRMS (ESI(+)) calcd for C21H19N2OS (M + H):
347.1218. Found: 347.1216.
N-(4-(2,4-Dimethylphenyl)thiazol-2-yl)-N-(prop-2-yn-1-yl)-
benzamide (6). 1H NMR (300 MHz, CDCl3): 8.36 (dd, J = 1.5 and
8.1 Hz, 2H), 7.52−7.43 (m, 4H), 7.20 (s, 1H), 7.18 (s, 2H), 4.75 (m,
2H), 2.42 (s, 3H), 2.22(m, 1H), 2.21 (s, 3H). 13C NMR (75 MHz,
CDCl3): 174.7, 167.3, 141.0, 138.5, 136.2, 135.9, 131.9, 131.6, 130.5,
129.5, 128.1, 127.3, 124.8, 98.0, 76.97, 72.7, 37.1, 21.5, 19.6. MS (ESI):
347 (M + H). HRMS (ESI(+)) calcd for C21H19N2OS (M + H):
347.1218. Found: 347.1210.
Methylation of 10 To Afford N-(4-(2,4-Dimethylphenyl)-3-
methylthiazol-2(3H)-ylidene)benzenesulfonamide (11) and N-
(4-(2,4-Dimethylphenyl)thiazol-2-yl)-N-methylbenzene-
sulfonamide (12). To a cooled mixture of NaH (0.07 g, 60% in oil,
1.6 mmol) in THF (5 mL) was added a solution of compound 10
(0.34g, 1 mmol) in THF (5 mL) dropwise. The mixture was warmed
to room temperature and stirred for 20 min. After that, the mixture
was cooled to 0 °C again and MeI (0.25 mL, 4.0 mmol) was added
dropwise. The mixture was then warmed to room temperature and
stirred for 2 h. Water (3 mL) was added to quench the reaction, and
the mixture was further diluted with water (10 mL). The mixture was
extracted with CH2Cl2 (3 × 10 mL). The combined organic phase was
dried with anhydrous MgSO4. After removal of all the solvent, the
residue was purified by silica gel chromatography (hexane/EtOAc =
4:1) to afford products 11 (more polar) (0.27 g, 75% yield) and 12
(0.034 g, 9% yield) as solids.
N-(4-Phenyl-3-ethylthiazol-2(3H)-ylidene)benzamide (7a).
1H NMR (300 MHz, CDCl3): 8.37 (dd, J = 1.5 and 7.5 Hz, 2H),
7.51−7.40 (m, 8H), 6.52 (s, 1H), 4.26 (q, J = 6.9 Hz, 2H), 1.34 (t, J =
6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3): 174.2, 168.1, 139.1, 137.1,
130.9, 129.6, 129.4, 129.2, 128.9, 128.0, 107.2, 42.5, 14.0. GC−MS:
308 (M+). HRMS (ESI(+)) calcd for C18H17N2OS (M + H):
309.1062. Found: 309.1052.
1
11: H NMR (300 MHz, CDCl3): 8.05−8.02 (m, 2H), 7.58−7.43
(m, 3H), 7.12−7.01 (m, 3H), 6.27 (s, 1H), 3.19 (s, 3H), 2.36 (s, 3H),
2.11 (s, 3H). 13C NMR (75 MHz, CDCl3): 166.8, 142.3, 140.6, 139.1,
137.6, 131.9, 131.4, 130.5, 128.6, 127.1, 126.6, 126.4, 103.5, 33.6, 21.3,
19.5. GC−MS: 358 (M+). HRMS (ESI(+)) calcd for C18H19N2O2S2
(M + H): 359.0888. Found: 359.0879.
N-(4-Phenyl-3-propylthiazol-2(3H)-ylidene)benzamide (7b).
1H NMR (300 MHz, CDCl3): 8.36 (dd, J = 1.2 and 7.5 Hz, 2H),
7.50−7.39 (m, 8H), 6.52 (s, 1H), 4.19 (q, J = 7.5 Hz, 2H), 1.76 (m,
2H), 0.83 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): 174.1,
168.5, 139.3, 137.2, 131.3, 130.9, 129.6, 129.5, 129.2, 128.8, 128.7,
128.0, 126.0, 107.2, 48.8, 21.9, 11.1. GC−MS: 322 (M+). HRMS
(ESI(+)) calcd for C19H19N2OS (M + H): 323.1218. Found:
323.1210.
1
12: H NMR (300 MHz, CDCl3): 7.83 (d, J = 7.2 Hz, 2H), 7.58−
7.38 (m, 4H), 7.02−6.98 (m, 2H), 6.88 (s, 1H), 3.45(s, 3H), 2.32 and
2.30 (ds, 6H). 13C NMR (75 MHz, CDCl3): 160.0, 151.2, 137.9,
136.8, 135.9, 133.8, 131.8, 131.4, 129.4, 129.3, 127.4, 126.6, 112.0,
36.7, 21.2, 21.1. GC−MS: 358 (M+). HRMS (ESI(+)) calcd for
C18H19N2O2S2 (M + H): 359.0888. Found: 359.0880.
1
N-(3-Allyl-4-phenylthiazol-2(3H)-ylidene)benzamide (7c). H
NMR (300 MHz, CDCl3): 8.35 (dd, J = 1.8 and 6.3 Hz, 2H), 7.49−
7.43 (m, 8H), 6.55 (s, 1H), 5.98 (m, 1H), 5.19 (d, J = 10.2 Hz, 1H),
4.98 (d, J = 17.4 Hz, 1H), 4.82 (d, J = 5.1 Hz, 2H). 13C NMR (75
MHz, CDCl3): 174.3, 168.5, 139.3, 137.0, 131.9, 131.4, 130.6, 129.7,
129.6, 129.2, 128.7, 128.0, 118.0, 107.1, 49.6. GC−MS: 320 (M+).
HRMS (ESI(+)) calcd for C19H17N2OS (M + H): 321.1062. Found:
321.1054.
General Procedure for the Acylation of 13 To Afford 14. To a
solution of 13 (0.01 mol) and DMAP (1.24 g, 0.01 mol) in anhydrous
dichloromethane was added the acyl chloride in dichloromethane
dropwise at 0 °C. After being stirred at room temperature for 2 h
under nitrogen atmosphere, the reaction mixture was concentrated in
vacuo. The saturated Na2CO3 solution was added to quench the
reaction, and the solution was extracted with ethyl acetate, dried over
MgSO4, and concentrated in vacuo. The residue was purified by flash
chromatography to give product 14 as a solid.
N-(4-Phenyl-3-(prop-2-yn-1-yl)thiazol-2(3H)-ylidene)-
1
benzamide (7d). H NMR (300 MHz, CDCl3): 8.40 (d, J = 6.6 Hz,
2H), 7.57−7.44 (m, 8H), 6.56 (s, 1H), 4.92 (d, J = 2.1 Hz, 2H). 13C
NMR (75 MHz, CDCl3): 174.4, 168.5, 138.6, 136.8, 131.6, 130.1,
129.9, 129.44, 129.37, 129.0, 128.1, 77.8, 72.8, 36.9. LC−MS (ESI):
319 (M + H). HRMS (ESI(+)) calcd for C19H15N2OS (M + H):
319.0905. Found: 319.0897.
N-(4-Methylthiazol-2-yl)benzamide (14a). 1H NMR (300
MHz, CDCl3): 7.99 (dd, J = 1.5 and 8.4 Hz, 2H), 7.52−7.47 (m,
3H), 6.55 (d, J = 0.9 Hz, 1H), 2.11 (s, 3H). 13C NMR (75 MHz,
CDCl3): 172.0, 165.3, 159.3, 146.8, 133.1, 132.8, 132.3, 130.0, 128.9,
128.3, 127.8, 108.4, 16.4. GC−MS: 218 (M+). HRMS (ESI(+)) calcd
for C11H11N2OS (M + H): 219.0592. Found: 219.0589.
N-Ethyl-N-(4-phenylthiazol-2-yl)benzamide (8a). 1H NMR
(300 MHz, CDCl3): 7.92 (m, 2H), 7.55−7.48 (m, 5H), 7.42 (m,
2H), 7.34 (m, 1H), 7.25 (s, 1H), 4.27 (q, J = 7.5 Hz, 2H), 1.38 (t, J =
6663
dx.doi.org/10.1021/jm500724x | J. Med. Chem. 2014, 57, 6653−6667

Journal of Medicinal Chemistry
Article
N-(4-(2-Chlorophenyl)thiazol-2-yl)benzamide (14b). 1H
NMR (300 MHz, CDCl3): 11.5 (bs, 1H), 7.75 (dd, J = 1.2 and 8.4
Hz, 2H), 7.63 (dd, J = 2.1 and 7.2 Hz, 1H), 7.51−7.45 (m, 2H), 7.38−
7.28 (m, 3H), 7.19−7.08 (m, 2H). 13C NMR (75 MHz, CDCl3):
165.3, 158.5, 146.3, 132.8, 132.7, 132.0, 131.7, 130.8, 130.4, 128.9,
128.7, 127.4, 126.8, 113.2. GC−MS: 314 (M+). HRMS (ESI(+)) calcd
for C16H12ClN2OS (M + H): 315.0359. Found: 315.0351.
4-Chloro-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
benzamide (14l). 1H NMR (300 MHz, CDCl3): 11.43 (bs, 1H), 7.73
(m, 2H), 7.30 (m, 2H), 7.12 (s, 1H), 6.88 (s, 2H), 3.85 (s, 6H),
3.83(s, 3H). 13C NMR (75 MHz, CDCl3): 164.4, 159.0, 153.3, 150.1,
139.1, 138.0, 130.3, 129.7, 129.0, 128.8, 107.9, 103.3, 60.8, 56.0. GC−
MS: 404 (M+). HRMS (ESI(+)) calcd for C19H18ClN2O4S (M + H):
405.0676. Found: 405.0664.
N-(4-(3-Bromophenyl)thiazol-2-yl)benzamide (14c). 1H NMR
(300 MHz, CDCl3): 11.68 (bs, 1H), 8.17−7.15 (m, 10H). 13C NMR
(75 MHz, CDCl3): 171.3, 165.3, 160.1, 148.3, 135.9, 133.5, 133.1,
133.0, 131.7, 131.1, 130.3, 130.2, 130.1, 129.5, 129.3, 128.9, 128.4,
127.8, 124.9, 122.9, 109.3. GC−MS: 360, 358 (M+). HRMS (ESI(+))
calcd for C16H12BrN2OS (M + H): 358.9854. Found: 358.9847.
N-(4-(4-Bromophenyl)thiazol-2-yl)benzamide (14d). 1H
NMR (300 MHz, CDCl3): 10.34 (bs, 1H), 7.90 (m, 2H), 7.67−7.41
(m, 7H), 7.20 (s, 1H). 13C NMR (75 MHz, CDCl3): 164.9, 158.7,
149.1, 133.1, 133.0, 131.83, 131.79, 129.0, 127.6, 127.4, 122.1, 108.5.
GC−MS: 358, 360 (M+). HRMS (ESI(+)) calcd for C16H12BrN2OS
(M + H): 358.9854. Found: 358.9848.
3,4-Dimethoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14m). H NMR (300 MHz, CDCl3): 11.07 (bs, 1H),
7.44 (dd, J = 1.8 and 8.1 Hz, 1H), 7.39 (s, 1H), 7.11 (s, 1H), 6.92 (s,
2H), 6.78 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 6H), 3.83(s, 3H),
3.82 (s, 3H). 13C NMR (75 MHz, CDCl3): 164.8, 159.2, 153.3, 152.8,
150.1, 148.9, 138.0, 129.9, 124.2, 120.8, 110.4, 110.3, 107.6, 103.3,
60.9, 56.0, 55.8. GC−MS: 430 (M+). HRMS (ESI(+)) calcd for
C21H23N2O6S (M + H): 431.1277. Found: 431.1265.
2,4-Dimethoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14n). H NMR (300 MHz, CDCl3): 11.03 (bs, 1H),
8.28 (d, J = 9.0 Hz, 1H), 7.10 (s, 2H), 7.09 (s, 1H), 6.68 (dd, J = 2.1
and 9.0 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 4.11 (s, 3H), 3.96(s, 6H),
3.90 (s, 3H), 3.89 (s, 3H). 13C NMR (75 MHz, CDCl3): 164.8, 162.6,
159.2, 158.1, 153.4, 149.9, 138.0, 134.5, 130.4, 112.0, 107.5, 106.1,
103.4, 98.7, 60.9, 56.4, 56.2, 55.7. HRMS (ESI(+)) calcd for
C21H23N2O6S (M + H): 431.1277. Found: 431.1270.
3,4,5-Trimethoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-
yl)benzamide (14o). 1H NMR (300 MHz, CDCl3): 11.50 (bs, 1H),
7.13 (d, J = 0.3 Hz, 1H), 7.06 (s, 2H), 6.90 (s, 2H), 3.87 (s, 3H),
3.84(s, 6H), 3.82 (s, 3H), 3.78 (s, 6H). 13C NMR (75 MHz, CDCl3):
165.1, 159.3, 153.3, 153.1, 150.2, 141.9, 138.1, 129.6, 126.8, 107.8,
104.8, 103.2, 60.9, 60.8, 56.1, 56.0. HRMS (ESI(+)) calcd for
C22H25N2O7S (M + H): 461.1382. Found: 461.1374.
N-(4-(3-Chlorophenyl)thiazol-2-yl)benzamide (14e). 1H NMR
(300 MHz, CDCl3): 10.19 (bs, 1H), 7.90 (m, 2H), 7.77 (t, J = 1.8 Hz,
1H), 7.66−7.56 (m, 2H), 7.49−7.44 (m, 1H), 7.32−7.26 (m, 2H),
7.20 (s, 1H). 13C NMR (75 MHz, CDCl3): 164.8, 158.5, 148.7, 135.9,
134.7, 133.0, 131.8, 130.0, 129.0, 128.0, 127.4, 126.2, 124.1, 109.1.
GC−MS: 314 (M+). HRMS (ESI(+)) calcd for C16H12ClN2OS (M +
H): 315.0359. Found: 315.0353.
N-(4-(3,5-Bis(trifluoromethyl)phenyl)thiazol-2-yl)benzamide
1
(14f). H NMR (300 MHz, CDCl3): 10.08 (bs, 1H), 8.24 (s, 2H),
7.93(m, 2H), 7.77 (s, 1H), 7.60 (m, 1H), 7.51−7.45 (m, 2H), 7.39 (s,
1H). 13C NMR (75 MHz, CDCl3): 165.0, 159.1, 147.3, 136.3, 133.4,
132.5, 132.0, 131.7, 129.3, 127.6, 126.2, 125.3, 121.7, 110.9. GC−MS:
416 (M+). HRMS (ESI(+)) calcd for C18H11F6N2OS (M + H):
417.0496. Found: 417.0486.
2,3-Dimethoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14p). H NMR (300 MHz, CDCl3): 11.38 (bs, 1H),
7.84 (dd, J = 1.5 and 7.8 Hz, 1H), 7.24−7.15 (m, 3H), 7.12 (s, 2H),
4.11 (s, 3H), 3.96(s, 6H), 3.92 (s, 3H), 3.89 (s, 3H). 13C NMR (75
MHz, CDCl3): 162.6, 157.5, 153.4, 152.6, 150.1, 148.0, 138.1, 130.3,
124.8, 124.0, 123.0, 117.0, 107.6, 103.4, 61.9, 60.9, 56.2. HRMS
(ESI(+)) calcd for C21H23N2O6S (M + H): 431.1277. Found:
431.1268.
N-(4-(3,4,5-Trimethoxyphenyl)thiazol-2-yl)benzamide (14g).
1
Yield: 1.9 g, 51%. H NMR (300 Hz, CDCl3): 10.10 (1H, bs), 7.93
(2H, J = 7.2 Hz, d), 7.59 (1H, m), 7.52−7.47 (2H, m), 7.13 (1H, s),
7.04 (2H, s), 3.92 (6H, s), 3.87 (3H,s). 13C NMR (75 Hz, CDCl3):
164.6, 158.1, 153.4, 150.0, 138.1, 133.0, 131.8, 130.0, 129.0, 127.3,
107.6, 103.3, 61.0, 56.2. MS-EI: 370 (M+). HRMS (ESI(+)) calcd for
C19H19N2O4S (M + H): 371.1066. Found: 371.1052. HRMS (ESI(+))
calcd for C19H19N2O4S (M + H): 371.1066. Found: 371.1060.
N-(5-Methyl-4-phenylthiazol-2-yl)benzamide (14h). 1H NMR
(300 MHz, CDCl3): 8.20 (d, J = 6.9 Hz, 2H), 8.09 (d, J = 7.2 Hz, 2H),
7.62−7.43 (m, 6H), 2.51 (s, 2H). 13C NMR (75 MHz, CDCl3): 171.3,
165.2, 157.1, 144.4, 133.7, 133.3, 132.8, 131.9, 130.3, 130.1, 128.8,
128.4, 128.3, 128.1, 127.9, 122.0, 12.0. GC−MS: 294 (M+). HRMS
(ESI(+)) calcd for C17H15N2OS (M + H): 295.0905. Found:
295.0897.
2-Methoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14q). H NMR (300 MHz, CDCl3): 11.19 (bs, 1H),
8.33 (dd, J = 1.5 and 7.8 Hz, 1H), 7.57 (dt, J = 1.5 and Hz, 1H), 7.19−
7.07 (m, 5H), 4.14 (s, 3H), 3.96 (s, 6H), 3.89 (s, 3H). 13C NMR (75
MHz, CDCl3): 162.7, 157.8, 157.7, 153.4, 150.0, 138.0, 134.5, 132.6,
130.3, 121.7, 119.0, 111.6, 107.7, 103.4, 60.9, 56.4, 56.2. HRMS
(ESI(+)) calcd for C20H21N2O5S (M + H): 401.1171. Found:
401.1165.
3-Methoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14r). H NMR (300 MHz, CDCl3): 10.75 (bs, 1H),
7.41−7.39 (m, 2H), 7.31 (m, 1H), 7.12 (s, 1H), 7.05 (m, 1H), 6.96 (s,
2H), 3.88 (s, 6H), 3.84 (s, 3H), 3.77 (s, 3H). 13C NMR (75 MHz,
CDCl3): 164.8, 159.8, 158.5, 153.3, 150.1, 138.0, 133.1, 129.9, 129.8,
119.4, 119.2, 112.2, 107.6, 103.3, 60.9, 56.0, 55.3. HRMS (ESI(+))
calcd for C20H21N2O5S (M + H): 401.1171. Found: 401.1164.
General Procedure for Methylation of 14. To a cooled mixture
of NaH (0.26 g, 60% in oil, 6.5 mmol) in THF (20 mL) was added a
solution of compound 14 (5 mmol) in THF (10 mL) dropwise. The
mixture was warmed to room temperature and stirred for 20 min. After
that, the mixture was cooled to 0 °C again and MeI (6.5 mmol) was
added dropwise. The mixture was then warmed to room temperature
and stirred for 2 h. Water (5 mL) was added to quench the reaction,
and the mixture was further diluted with water (50 mL). The mixture
was extracted with CH2Cl2 (3 × 50 mL). The combined organic phase
was dried with anhydrous MgSO4. After removal of all the solvent, the
residue was purified by silica gel chromatography (hexane/EtOAc =
4:1) to afford products 15 (more polar) and 16 as solids.
N-(4-(4-Fluorophenyl)-5-methylthiazol-2-yl)benzamide
1
(14i). H NMR (300 MHz, CDCl3): 10.6 (bs, 1H), 7.82 (dd, J = 1.2
and 8.1 Hz, 2H), 7.53−7.41 (m, 5H), 7.01 (m, 2H). 13C NMR (75
MHz, CDCl3): 164.7, 155.1, 144.2, 132.7, 132.0, 130.7, 130.0, 129.9,
128.8, 127.4, 122.2, 115.4, 115.2, 12.1. GC−MS: 312 (M+). HRMS
(ESI(+)) calcd for C17H14FN2OS (M + H): 313.0811. Found:
313.0802.
N-(5-(p-Tolyl)thiazol-2-yl)benzamide (14j). 1H NMR (300
MHz, CDCl3): 10.01 (bs, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.69 (d, J
= 8.1 Hz, 2H), 7.57−7.45 (m, 3H), 7.19 (d, J = 8.1 Hz, 2H), 7.14 (s,
1H), 2.37 (s, 3H). 13C NMR (75 MHz, CDCl3): 165.2, 158.9, 150.3,
137.9, 132.7, 131.9, 131.4, 129.4, 128.7, 127.4, 126.0, 107.3, 21.2. GC−
MS: 294 (M+). HRMS (ESI(+)) calcd for C17H15N2OS (M + H):
295.0905. Found: 295.0899.
4-Methoxy-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
1
benzamide (14k). H NMR (300 MHz, CDCl3): 10.92 (bs, 1H),
N-(3,4-Dimethylthiazol-2(3H)-ylidene)benzamide (15a). 1H
NMR (300 MHz, CDCl3): 7.83 (dd, J = 1.8 and 7.8 Hz, 2H),
7.47−7.43 (m, 3H), 6.29 (d, J = 1.2 Hz, 1H), 3.79 (s, 3H), 2.31 (d, J =
1.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): 174.0, 169.0, 137.1, 134.4,
131.3, 129.2, 128.0, 104.3, 32.9, 14.4. GC−MS: 232 (M+). HRMS
7.80 (d, J = 8.7 Hz, 2H), 7.10 (s, 1H), 6.94 (s, 2H), 6.84 (d, J = 8.7
Hz, 2H), 3.86 (s, 6H), 3.84(s, 3H), 3.83 (s, 3H). 13C NMR (75 MHz,
CDCl3): 164.6, 163.1, 159.1, 153.2, 149.9, 137.9, 130.0, 129.4, 124.0,
114.0, 107.5, 103.3, 60.8, 56.0, 55.4. HRMS (ESI(+)) calcd for
C20H21N2O5S (M + H): 401.1171. Found: 401.1165.
6664
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(ESI(+)) calcd for C12H13N2OS (M + H): 233.0749. Found:
233.0741.
NMR (75 MHz, CDCl3): 173.9, 168.4, 162.4, 153.5, 139.2, 139.1,
131.1, 129.7, 126.0, 113.2, 106.6, 106.4, 61.0, 56.3, 55.3, 34.9, 29.7.
HRMS (ESI(+)) calcd for C21H23N2O5S (M + H): 415.1328. Found:
415.1321.
4-Chloro-N-(3-methyl-4-(3,4,5-trimethoxyphenyl)thiazol-
2(3H)-ylidene)benzamide (15l). 1H NMR (300 MHz, CDCl3):
8.30 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.7 Hz, 2H), 6.60 (s, 2H), 6.58
(s, 1H), 3.93 (s, 3H), 3.90 (s, 6H), 3.75 (s, 3H). 13C NMR (75 MHz,
CDCl3): 173.2, 168.8, 153.6, 139.5, 139.3, 137.6, 135.5, 130.7, 128.3,
125.7, 106.8, 106.7, 61.0, 56.4, 35.0. HRMS (ESI(+)) calcd for
C20H20ClN2O4S (M + H): 419.0832. Found: 419.0824.
N-(4-(2-Chlorophenyl)-3-methylthiazol-2(3H)-ylidene)-
1
benzamide (15b). H NMR (300 MHz, CDCl3): 8.38 (dd, J = 1.8
and 7.8 Hz, 2H), 7.54−7.39 (m, 7H), 6.60 (s, 1H), 3.63 (s, 3H). 13C
NMR (75 MHz, CDCl3): 174.3, 168.4, 136.9, 136.3, 134.8, 132.3,
131.53, 131.49, 130.0, 129.9, 129.2, 128.1, 127.3, 108.1, 34.0. GC−MS:
328 (M+). HRMS (ESI(+)) calcd for C17H14ClN2OS (M + H):
329.0515. Found: 329.0505.
N-(4-(3-Bromophenyl)-3-methylthiazol-2(3H)-ylidene)-
1
benzamide (15c). H NMR (300 MHz, CDCl3): 7.83 (dd, J = 1.8
and 8.1 Hz, 2H), 7.64 (m, 1H), 7.59 (m, 1H), 7.51−7.36 (m, 6H),
6.60 (s, 1H), 3.75 (s, 3H). 13C NMR (75 MHz, CDCl3): 174.4, 168.9,
137.8, 136.8, 132.8, 132.5, 132.2, 131.6, 130.5, 129.3, 128.1, 127.8,
123.0, 107.8, 34.9. GC−MS: 374, 372 (M+). HRMS (ESI(+)) calcd for
C17H14BrN2OS (M + H): 373.0010. Found: 373.0006.
3-Methoxy-N-(3-methyl-4-(3,4,5-trimethoxyphenyl)thiazol-
1
2(3H)-ylidene)benzamide (15m). H NMR (300 MHz, CDCl3):
8.00 (m, 1H), 7.91 (m, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.05 (m, 1H),
6.60 (s, 2H), 6.57 (s, 1H), 3.93 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H),
3.76 (s, 3H). 13C NMR (75 MHz, CDCl3): 174.1, 168.8, 159.5, 153.5,
139.4, 139.2, 138.5, 129.0, 125.8, 121.8, 118.0, 113.6, 106.7, 106.6,
61.0, 56.4, 55.4, 35.0. HRMS (ESI(+)) calcd for C21H23N2O5S (M +
H): 415.1328. Found: 415.1320.
N-(4-(4-Bromophenyl)-3-methylthiazol-2(3H)-ylidene)-
1
benzamide (15d). H NMR (300 MHz, CDCl3): 8.37 (dd, J = 1.5
and 8.1 Hz, 2H), 7.65 (d, J = 6.6 Hz, 2H), 7.51−7.43 (m, 4H), 7.28 (d,
J = 7.2 Hz, 1H), 6.58 (d, J = 0.9 Hz, 1H), 3.74 (d, J = 2.1 Hz, 3H). 13C
NMR (75 MHz, CDCl3): 174.3, 168.9, 138.2, 136.9, 132.3, 131.9,
131.6, 130.8, 129.5, 128.1, 124.2, 107.4, 34.9. GC−MS: 372, 374 (M+).
HRMS (ESI(+)) calcd for C17H14BrN2OS (M + H): 373.0010. Found:
373.0000.
N-(4-(3-Chlorophenyl)-3-methylthiazol-2(3H)-ylidene)-
benzamide (15e). 1H NMR (300 MHz, CDCl3): 8.37 (d, J = 7.8 Hz,
2H), 7.51−7.43 (m, 6H), 7.32 (m, 1H), 6.59(s, 1H), 3.75 (s, 3H). 13C
NMR (75 MHz, CDCl3): 174.4, 168.9, 137.9, 136.8, 135.0, 132.3,
131.6, 130.3, 129.9, 129.4, 129.3, 128.1, 127.4, 107.8, 34.9. GC−MS:
328 (M+). HRMS (ESI(+)) calcd for C17H14ClN2OS (M + H):
329.0515. Found: 329.0508.
N-(4-(3,5-Bis(trifluoromethyl)phenyl)-3-methylthiazol-2(3H)-
ylidene)benzamide (15f). 1H NMR (300 MHz, CDCl3): 8.37 (dd, J
= 1.5 and 8.1 Hz, 2H), 8.03 (s, 1H), 7.91 (s, 2H), 7.50−7.46 (m, 3H),
6.73 (s, 1H), 3.77 (s, 3H). 13C NMR (75 MHz, CDCl3): 174.4, 168.9,
136.4, 136.0, 132.8, 132.7, 132.4, 131.7, 129.2, 128.0, 123.3, 120.9,
109.4, 34.8. GC−MS: 430 (M+). HRMS (ESI(+)) calcd for
C19H13F6N2OS (M + H): 431.0653. Found: 431.0646.
N-(4-Cyclopropyl-3-methylthiazol-2(3H)-ylidene)benzamide
1
(15n). H NMR (300 MHz, CDCl3): 8.35 (dd, J = 1.8 and 7.8 Hz,
2H), 7.49−7.41 (m, 3H), 6.23 (d, J = 1.2 Hz, 1H), 3.95 (s, 3H), 1.75
(m, 1H), 1.02 (m, 2H), 0.73 (m, 2H). 13C NMR (75 MHz, CDCl3):
174.0, 169.1, 140.6, 137.1, 131.3, 129.2, 128.0, 103.9, 33.2, 8.7, 5.9.
GC−MS: 258 (M+). HRMS (ESI(+)) calcd for C14H15N2OS (M +
H): 259.0509. Found: 259.0519.
3,4,5-Trimethoxy-N-(3-methyl-4-(3,4,5-trimethoxyphenyl)-
thiazol-2(3H)-ylidene)benzamide (15o). 1H NMR (300 MHz,
CDCl3): 7.68 (s, 2H), 6.61 (s, 2H), 6.57 (s, 1H), 3.96 (s, 6H), 3.93 (s,
3H), 3.92(s, 3H), 3.91 (s, 6H), 3.77 (s, 3H). 13C NMR (75 MHz,
CDCl3): 173.7, 168.8, 153.5, 152.7, 141.1, 139.4, 139.2, 132.3, 125.8,
106.7, 106.6, 106.4, 61.0, 60.9, 56.3, 56.1, 34.9. HRMS (ESI(+)) calcd
for C23H27N2O7S (M + H): 475.1539. Found: 475.1526.
N-(4-(2-Chlorophenyl)thiazol-2-yl)-N-methylbenzamide
1
(16a). H NMR (300 MHz, CDCl3): 7.97 (dd, J = 1.8 and 7.8 Hz,
1H), 7.60−7.46 (m, 7H), 7.37−7.23 (m, 2H), 3.74 (s, 3H). 13C NMR
(75 MHz, CDCl3): 170.4, 159.0, 146.0, 134.5, 133.5, 132.1, 131.3,
130.9, 130.6, 128.73, 128.66, 127.6, 126.9, 114.6, 38.5. GC−MS: 328
(M+). HRMS (ESI(+)) calcd for C17H14ClN2OS (M + H): 329.0515.
Found: 329.0515.
N-(4-(3,4,5-Trimethoxyphenyl)-3-methylthiazol-2(3H)-
1
yliene)benzamide (15g). Yield: 0.69 g, 36%. H NMR (300 Hz,
CDCl3): 8.37 (2H, J = 7.8 Hz, d), 7.50−7.43 (3H, m), 6.60 (2H, s),
6.56 (1H, s), 3.92 (3H, s), 3.90 (6H, s), 3.76 (3H, s). 13C NMR (75
Hz, CDCl3): 174.3, 168.7, 153.5, 139.4, 139.1, 137.0, 131.5, 129.2,
128.1, 125.9, 106.7, 106.6, 61.0, 56.4, 35.0. MS-EI: 384 (M+). HRMS
(ESI(+)) calcd for C20H21N2O4S (M + H): 385.1222. Found:
385.1215.
N-(4-(3-Bromophenyl)thiazol-2-yl)-N-methylbenzamide
(16b). 1H NMR (300 MHz, CDCl3): 8.1 (s, 1H), 7.83 (d, J = 7.8 Hz,
1H), 7.59−7.44 (m, 6H), 7.31−7.28 (m, 2H), 3.76 (s, 3H). 13C NMR
(75 MHz, CDCl3): 170.5, 160.2, 147.9, 136.7, 134.4, 131.0, 130.7,
130.2, 129.1, 128.7, 127.6, 124.5, 122.9, 110.2, 38.5. GC−MS: 374, 372
(M+). HRMS (ESI(+)) calcd for C17H14BrN2OS (M + H): 373.0010.
Found: 373.0001.
N-(3,5-Dimethyl-4-phenylthiazol-2(3H)-ylidene)benzamide
1
N-(4-(4-Bromophenyl)thiazol-2-yl)-N-methylbenzamide
(15h). H NMR (300 MHz, CDCl3): 8.37 (dd, J = 1.8 and 8.1 Hz,
1
(16c). H NMR (300 MHz, CDCl3): 7.80 (d, J = 8.7 Hz, 2H), 7.59−
2H), 7.53−7.42 (m, 6H), 7.33−7.30 (m, 2H), 3.61 (s, 3H), 2.17 (s,
3H). 13C NMR (75 MHz, CDCl3): 174.0, 167.0, 137.2, 134.1, 131.3,
130.4, 129.8, 129.5, 129.2, 129.1, 128.0, 117.8, 35.0. GC−MS: 308
(M+). HRMS (ESI(+)) calcd for C18H17N2OS (M + H): 309.1062.
Found: 309.1055.
7.50 (m, 7H), 3.75 (s, 3H). 13C NMR (75 MHz, CDCl3): 170.4,
160.2, 148.3, 134.5, 133.7, 131.8, 131.0, 128.7, 127.6, 121.8, 109.6,
38.5. GC−MS: 372, 374 (M+). HRMS (ESI(+)) calcd for
C17H14BrN2OS (M + H): 373.0010. Found: 373.0005.
N-Methyl-N-(4-(3,4,5-trimethoxyphenyl)thiazol-2-yl)-
benzamide (16d). Yield: 0.35 g, 18%. 1H NMR (CDCl3): 7.61−7.57
(2H, m), 7.55−7.50 (3H, m), 7.19 (1H, s), 7.15 (2H, s), 3.95 (6H, s),
3.89 (3H, s), 3.77 (3H, s). 13C NMR (CDCl3): 170.4, 160.0, 153.5,
149.5, 138.3, 134.5, 131.0, 130.5, 128.7, 127.6, 108.9, 103.4, 61.0, 56.2,
38.5. MS-EI: 384 (M+). HRMS (ESI(+)) calcd for C20H21N2O4S (M +
H): 385.1222. Found: 385.1216.
N-(4-(4-Fluorophenyl)-3,5-dimethylthiazol-2(3H)-ylidene)-
1
benzamide (15i). H NMR (300 MHz, CDCl3): 8.36 (dd, J = 0.9
and 7.8 Hz, 2H), 7.47−7.44 (m, 3H), 7.32−7.22 (m, 4H), 3.60 (s,
3H), 2.15 (s, 3H). 13C NMR (75 MHz, CDCl3): 174. 0, 167.0, 164.9,
161.6, 137.1, 133.0, 132.4, 132.2, 131.4, 129.2, 128.0, 125.74, 125.70,
118.2, 116.5, 116.2, 34.9, 12.2. GC−MS: 326 (M+). HRMS (ESI(+))
calcd for C18H16FN2OS (M + H): 327.0967. Found: 327.0957.
N-(3-Methyl-5-(p-toyl)thiazol-2(3H)-ylidene)benzamide
N-(4-(4-Fluorophenyl)-5-methylthiazol-2-yl)-N-methylben-
1
1
zamide (16e). H NMR (300 MHz, CDCl3): 7.69−7.64 (m, 2H),
(15j). H NMR (300 MHz, CDCl3): 8.38 (dd, J = 1.5 and 7.8 Hz,
7.57−7.48 (m, 5H), 7.16−7.10 (m, 2H), 3.67 (s, 3H), 2.52 (s, 3H).
13C NMR (75 MHz, CDCl3): 170.2, 163.7, 160.5, 156.2, 143.8, 134.6,
131.6, 130.8, 130.1, 130.0, 128.6, 127.6, 123.3, 115.4, 115.1, 37.9, 12.1.
GC−MS: 326 (M+). HRMS (ESI(+)) calcd for C18H16FN2OS (M +
H): 327.0967. Found: 327.0959.
2H), 7.48−7.44 (m, 3H), 7.30 (s, 4H), 6.53 (s, 1H), 3.74 (s, 3H), 2.44
(s, 3H). 13C NMR (75 MHz, CDCl3): 174.2, 168.9, 139.8, 139.6,
137.1, 131.4, 129.6, 129.23, 129.17, 128.0, 127.7, 106.6, 34.9, 21.4.
GC−MS: 308 (M+). HRMS (ESI(+)) calcd for C18H17N2OS (M +
H): 309.1062. Found: 309.1054.
4-Methoxy-N-(3-methyl-4-(3,4,5-trimethoxyphenyl)thiazol-
2(3H)-ylidene)benzamide (15k). 1H NMR (300 MHz, CDCl3):
8.33 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.60 (s, 2H), 6.53
(s, 1H), 3.92 (s, 3H), 3.90(s, 6H), 3.88 (s, 3H), 3.74 (s, 3H). 13C
N-Methyl-N-(5-(p-tolyl)thiazol-2-yl)benzamide (16f). 1H
NMR (300 MHz, CDCl3): 7.82 (d, J = 8.1 Hz, 2H), 7.59−7.49 (m,
5H), 7.24−7.19 (m, 3H), 3.76 (s, 3H), 2.39 (s, 3H). 13C NMR (75
MHz, CDCl3): 170.4, 159.9, 149.5, 137.7, 134.7, 132.0, 130.8, 129.4,
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128.6, 127.6, 126.0, 108.4, 38.4, 21.3. GC−MS: 308 (M+). HRMS
(ESI(+)) calcd for C18H17N2OS (M + H): 309.1062. Found:
309.1055.
was used as a chemoattractant in the lower wells. After 24 h,
membranes were scrubbed, fixed with 10% phospho-buffered formalin,
permeabilized with 100% ice-cold methanol, and stained with 0.1%
crystal violet in 20% methanol. Membranes were removed and
mounted on glass slides for visualization by light microscopy. For dose
dependent migration assays, MDA-MB-231 cells were seeded in the
Transwell migration chambers as described above and treated with
10−8, 10−7, 10−6, and 10−5 M concentrations of each thiazole
compound for measurement of resulting cell migration; 0.1%
DMSO was used as vehicle control. Data are represented as % of
migrated cells treated with vehicle per 100× field of view (100×).
Cell Survival/Growth Assay. For growth assay in the presence of
10−5 M individual thiazole compound, MDA-MB-231 cells were plated
in six-well plates at a density of 50 000 per well in DMEM medium
supplemented with 10% FBS. The cells were then cultured for 5 days,
while equal treatment volumes of DMSO were used as vehicle control.
Cell numbers were counted with a Coulter instrument (Beckman-
Coulter). The ratio of thiazole compound treated cell numbers to
vehicle treated cell numbers was defined as survival ratio. Experiments
were conducted in triplicate and data represented as the mean SD.
Invasion Assays. Matrigel-coated invasion chambers (BD
Biosciences, NJ) were used to determine the inhibitory effect of the
thiazole derivatives on MDA-MB-231 cells. Briefly, cells in exponential
phase of growth were serum-starved for 24 h prior to seeding,
detached by brief trypsinization, and resuspended in medium
containing DMSO or thiazole derivatives. The Matrigel invasion
inserts were prepared following the manufacturer’s instructions. MDA-
MB-231 cells (2.5 × 104 cells/well) were seeded in the upper chamber,
and medium supplemented with 10% FBS as chemoattractant
containing DMSO or 10 μM thiazole derivatives was added to the
bottom well. After incubation for 24 h, the noninvasive cells were
removed from the upper surface of the membrane by “scrubbing”, and
the invasive cells on the under surface of the membrane were fixed
with 4% formaldehyde for 10 min at room temperature and stained
with 0.04% crystal violet, counted microscopically at 100×
magnification. Five fields per membrane were randomly selected and
counted in each group. The relative invasion of cells was calculated as
the percentage invasion through the Matrigel membrane relative to
that of DMSO treated cells.
Overexpression of Human Fascin 1. Construction of Fascin
Vector. The fascin total RNA samples were extracted from HeLa cells
using a PureLink total RNA purification system (Invitrogen) and
quantitatively analyzed using a Nanodrop spectrophotometer
(Thermo Scientific). The fascin cDNA was generated using a
Superscript III one-step RT-PCR system (Invitrogen) with the
following primers: fascin1-F (sense) 5′-GAA TTC ATG ACC GCC
AAC GGC ACA GC-3′ and fascin1-R (antisense) 5′-AAG CTT CTA
GTA CTC CCA GAG CGA GGC-3′. The RT-PCR reaction was
carried out as follows: step 1, 45 °C for 30 min and 94 °C for 2 min;
step 2, for 35 cycles 94 °C for 15 s, 51 °C for 30 s, and 72 °C for 1 min
and 30 s; step 3, 72 °C for 5 min and hold at 4 °C. Human fascin
cDNA (1.5 kilobases [kb]) samples were then cloned into a pcDNA
3.1 vector (Invitrogen).
Overexpression of Fascin in MDA-MB-231 Cell. For fascin
overepression, the MDA-MB-231 cells were seeded at a density of 5 ×
105 cells par well in six-well plates. At 70−80% confluence, the cells
were transiently tranfected with 4 μg of the fascin-expressing plasmid
pcDNA-fascin. The transfection was achieved by incubating the cells
overnight at 37 °C in a CO2 incubator with Lipofectamine 2000
reagent (Invitrogen). The next day, cell medium was replaced with
1000 μg/m G418 followed by medium replacement every 2−3 days
for a total 10−12 days. Stable clones were isolated after selection with
500 μg/mL G418 for 2−3 weeks, and fascin expression levels were
determined by Western blot.
4-Methoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)thiazol-
1
2-yl)benzamide (16g). H NMR (300 MHz, CDCl3): 7.58 (d, J =
8.4 Hz, 2H), 7.16 (s, 1H), 7.16 (s, 2H), 7.00 (d, J = 8.7 Hz, 2H), 3.95
(s, 6H), 3.89(s, 6H), 3.81 (s, 3H). 13C NMR (75 MHz, CDCl3):
170.2, 161.8, 160.4, 153.4, 149.2, 138.0, 130.6, 130.0, 126.4, 113.9,
108.7, 103.4, 60.9, 56.2, 55.4, 38.8, 29.7. HRMS (ESI(+)) calcd for
C21H23N2O5S (M + H): 415.1328. Found: 415.1317.
3,4-Dimethoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)-
thiazol-2-yl)benzamide (16h). 1H NMR (300 MHz, CDCl3):
7.23−7.17 (m, 3H), 7.16 (s, 2H), 6.95 (d, J = 8.4 Hz, 1H), 3.96 (s,
3H), 3.95(s, 6H), 3.93 (s, 3H), 3.88 (s, 3H), 3.82 (s, 3H). 13C NMR
(75 MHz, CDCl3): 170.1, 160.4, 153.4, 151.4, 149.3, 149.0, 138.1,
130.5, 126.5, 121.3, 111.3, 110.4, 108.7, 103.4, 61.0, 56.2, 56.0, 38.8,
29.7. GC−MS: 444 (M+). HRMS (ESI(+)) calcd for C22H25N2O6S
(M + H): 445.1433. Found: 445.1426.
2,4-Dimethoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)-
1
thiazol-2-yl)benzamide (16i). H NMR (300 MHz, CDCl3): 7.35
(d, J = 8.4 Hz, 1H), 7.16 (s, 3H), 6.60 (dd, J = 2.4 and 8.4 Hz, 1H),
6.52 (d, J = 2.1 Hz, 1H), 3.94(s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 3.84
(s, 3H), 3.64 (s, 3H). 13C NMR (75 MHz, CDCl3): 168.9, 162.8,
159.9, 157.1, 153.4, 149.0, 138.0, 130.7, 129.9, 117.2, 108.5, 105.0,
103.4, 98.5, 60.9, 56.2, 55.6, 55.5, 36.7. HRMS (ESI(+)) calcd for
C22H25N2O6S (M + H): 445.1433. Found: 445.1424.
3,4,5-Trimethoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)-
1
thiazol-2-yl)benzamide (16j). H NMR (300 MHz, CDCl3): 7.19
(s, 1H), 7.16 (s, 2H), 6.82 (s, 2H), 3.95 (s, 6H), 3.92 (s, 3H), 3.90 (s,
6H), 3.89(s, 3H), 3.80 (s, 3H). 13C NMR (75 MHz, CDCl3): 170.1,
160.2, 153.5, 153.3, 149.4, 140.3, 138.2, 130.5, 129.5, 108.9, 105.1,
103.4, 61.01, 60.98, 56.3, 56.2, 38.7. HRMS (ESI(+)) calcd for
C23H27N2O7S (M + H): 475.1539. Found: 475.1530.
2,3-Dimethoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)-
thiazol-2-yl)benzamide (16k). 1H NMR (300 MHz, CDCl3): 7.20−
7.18 (m, 2H), 7.15 (s, 2H), 7.05 (dd, J = 1.5 and 8.1 Hz, 1H), 6.93
(dd, J = 1.5 and 7.8 Hz, 1H), 3.95(s, 6H), 3.93 (s, 3H), 3.88 (s, 3H),
3.86 (s, 3H), 3.63 (s, 3H). 13C NMR (75 MHz, CDCl3): 168.4, 159.4,
153.4, 152.7, 149.1, 145.2, 138.0, 130.5, 130.0, 124.9, 119.0, 114.0,
108.7, 103.4, 61.7, 60.9, 56.2, 55.9, 36.7. HRMS (ESI(+)) calcd for
C22H25N2O6S (M + H): 445.1433. Found: 445.1428.
2-Methoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)thiazol-
1
2-yl)benzamide (16l). H NMR (300 MHz, CDCl3): 7.47 (m, 1H),
7.38 (dd, J = 1.8 and 7.5 Hz, 1H), 7.18 (s, 1H), 7.16 (s, 2H), 7.08 (dt,
J = 0.9 and 7.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 3.95 (s, 6H), 3.88 (s,
3H), 3.86 (s, 3H), 3.63 (s, 3H). 13C NMR (75 MHz, CDCl3): 168.9,
155.5, 153.4, 149.1, 138.0, 131.7, 130.6, 128.3, 124.5, 121.1, 111.0,
108.7, 103.4, 60.9, 56.2, 55.6, 36.5. HRMS (ESI(+)) calcd for
C21H23N2O5S (M + H): 415.1328. Found: 415.1319.
3-Methoxy-N-methyl-N-(4-(3,4,5-trimethoxyphenyl)thiazol-
2-yl)benzamide (16m). 1H NMR (300 MHz, CDCl3): 7.41 (m,
1H), 7.19 (s, 1H), 7.15 (s, 2H), 7.15−7.05 (m, 3H), 3.95 (s, 6H), 3.89
(s, 3H), 3.86 (s, 3H), 3.76 (s, 3H). 13C NMR (75 MHz, CDCl3):
170.2, 159.7, 153.4, 149.3, 138.1, 135.7, 130.5, 129.8, 119.5, 116.8,
112.8, 108.9, 103.4, 60.9, 56.2, 55.4, 38.4. HRMS (ESI(+)) calcd for
C21H23N2O5S (M + H): 415.1328. Found: 415.1322.
Cell Culture. MDA-MB-231 cell line was a gift from Dr. KiTani
Johnson, and HeLa cell line was a gift from Dr. Thomas Wiese, both of
Xavier University of Louisiana College of Pharmacy. A549 cells were
generously provided by Dr. Mandip Sachdeva of Florida A&M
University College of Pharmacy. These cancer cell lines were routinely
cultured in DMEM medium supplemented with 10% FBS, 4 mM
glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL
streptomycin, and 0.25 μg/mL amphotericin. Cultures were
maintained in 5% carbon dioxide at a temperature of 37 °C.
In Vitro Migration Assays. Migration assays were performed
following the manufacturer’s instructions (BD Falcon). Briefly, MDA-
MB-231, A549, or HeLa cells were seeded at a density of 2.5 × 104 in
500 μL of serum-free and phenol red-free media in the upper chamber
of a 24-well Transwell system. DMEM supplemented with FBS (5%)
Chick Embryo Chorioallantoic Membrane (CAM) Assay.
Fertilized embryos (Charles River Laboratories, Charleston, SC)
were incubated at 37.5 °C for 3 days, removed from their shell using a
Dremel tool, and placed into a covered weighing boat for 7 further
days of incubation. Solidified 30 μL onplants containing 2.1 mg/mL
rat tail collagen (BD Biosciences, Bedford, MA) and 10 ng of bFGF
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and 30 ng of VEGF in the presence or absence of thiazole analogues
5o and 5p were placed on the CAM over two pieces of nylon mesh
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by a second masked scorer) are presented as the mean standard
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AUTHOR INFORMATION
Corresponding Authors
*S.Z.: phone, 504-520-7824; e-mail, szheng@xula.edu.
*G.W.: phone, 504-520-5076; e-mail, gwang@xula.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH-NIMHD through Grant
8G12MD007595 and NIH-NIGMS through Grants
8P20GM103424 and P20RR016456 and in part by Louisiana
Cancer Research Consortium. We thank Thomas Vu, Bria
Carmichael, Briana Jarrett, Sydnie Turner, Eric Stewart, and
Elise LeMelle for technical assistance in the CAM assays.
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Neoplasia 2008, 10, 149−159.
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Fascin overexpression promotes neoplastic progression in oral
squamous cell carcinoma. BMC Cancer 2012, 12, 32.
ABBREVIATIONS USED
■
DMSO, dimethyl sulfoxide; CAM, chorioallantoic membrane;
FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle
medium; FITC, fluorescein isothiocyanate; PBS, phosphate
buffered saline; bFGF, basic fibroblast growth factor; VEGF,
vascular endothelial growth factor; BV, bFGF + VEGF
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H.; Kong, C. The role of fascin in migration and invasion of urothelial
carcinoma of the bladder. Urol. Int. 2013, 91, 227−235.
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proteins. FEBS Lett. 1986, 203, 139−143.
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