Efficient synthesis and cell migration inhibitory effect of substituted benzamidothiazolylpyrazole-capped AWD*I-NH2

Article abstract of DOI:10.1016/j.bmcl.2019.126914

Substituted (2-benzamidothiazol-5-yl)pyrazole-capped AWD*I-NH₂ were synthesized and their antimigration activity was studied. The improved efficiency and scalability of the analog synthesis was achieved via a late-stage diversification of the benzoyl group and a convergent route in which the bisazole capping agents and off-resin peptide AWD*I-NH₂ were prepared in parallel and coupled together in solution at the last step. Bioassay results indicate that all the peptidomimetics can significantly inhibit the migration of breast cancer cells MDA-MB-231 but possess no apparent cytotoxicity. In general, the antimigration potency of the peptidomimetics is correlated to the electron-withdrawing capacity of the substituents on the terminal phenyl ring. The inhibitory effect shows dose-dependent and holds also against lung and cervical cancer cells. The level of f-actin was reduced dramatically in cells treated with the inhibitor, suggesting that the migration inhibitory effect is related to the disruption of cell locomotive protrusions.

Full text of DOI:10.1016/j.bmcl.2019.126914

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis and cell migration inhibitory effect of substituted
benzamidothiazolylpyrazole-capped AWD*I-NH2
Mian Yanga,1, Jun Chena,1, Wancai Penga, Qiqi Lia, Hui Shaoa, Guanping Tanga,
⁎
⁎
Tong-Cun Zhangb, Yoshikazhu Takadac, Long Yea, , Xing-Hua Liaob,
a Department of Chemistry, School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan, Hubei 430081, PR China
b School of Life Sciences and Health, Wuhan University of Science and Technology, Wuhan, Hubei 430081, PR China
c Department of Dermatology, Biochemistry and Molecular Medicine, University of California Davis, School of Medicine, Sacramento, CA, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Substituted (2-benzamidothiazol-5-yl)pyrazole-capped AWD*I-NH2 were synthesized and their antimigration
activity was studied. The improved efficiency and scalability of the analog synthesis was achieved via a late-
stage diversification of the benzoyl group and a convergent route in which the bisazole capping agents and off-
resin peptide AWD*I-NH2 were prepared in parallel and coupled together in solution at the last step. Bioassay
results indicate that all the peptidomimetics can significantly inhibit the migration of breast cancer cells MDA-
MB-231 but possess no apparent cytotoxicity. In general, the antimigration potency of the peptidomimetics is
correlated to the electron-withdrawing capacity of the substituents on the terminal phenyl ring. The inhibitory
effect shows dose-dependent and holds also against lung and cervical cancer cells. The level of f-actin was
reduced dramatically in cells treated with the inhibitor, suggesting that the migration inhibitory effect is related
to the disruption of cell locomotive protrusions.
Cell migration
Inhibition
Peptidomimetics
Synthesis
f-Actin
The blockade of metastasis is crucial for the treatment of cancer. As
metastasis occurs at the late stage of cancer, tumor cells migrate and
invade from their primary site to patient’s other healthy tissues, causing
serious or even lethal detriment to the patient’s multiple organs and
also diminishing the opportunity for site-targeting treatments such as
surgical operation and radiotherapy etc. Indeed, nearly ninety percent
mortality of cancer patients is attributable to the metastatic spread of
the disease rather than to the primary tumor.1–3 Therefore an anti-
metastatic treatment could be a very useful disease-controlling strategy
as it could stop the spread of cancer cells, reduce tissue damage caused
by the invading cells and even lock the tumor at its primary site to allow
for a clean removal of the tumor.
forth.11–15 (+)-Migrastatin especially attracted a wide-spread interest
because further development based on its core macrocyclic structure
actually led to the invention of potent antimetastatic agents that are
effective in vivo.16–18 Encouraged by the progress in the area of mi-
gration inhibitors and good biocompatibility of peptidomimetics, we
did a further study on a hit compound 7a, which is a peptidomimetic
migration inhibitor and an antagonist against the protein–protein in-
teraction between lipase sPLA2-IIA and integrin αvβ3.19 The further
study indicates that 7a and its analogs can significantly inhibit not only
sPLA2-IIA-induced cell migration but also intrinsic migratory capacity
without effecting substantial cytotoxicity. In addition, this type of
peptidomimetics can also inhibit the migration of multiple other cancer
cell lines, showing a good potential as a broad-spectrum antimetastatic
therapeutic agent. Herein we report the details of these exciting results.
To access 7a and its analogs efficiently and at an increasable scale,
we introduced the diverse 2-benzamido groups onto the thiazole ring at
the late stage of the synthesis of the bisazole capping agent 6 and
coupled the agent with peptide AWD*I in solution (Scheme 1). To
achieve the late-stage diversification, the amino group of the starting
material (1) has to be protected first and deprotected later for amida-
tion after the pyrazole ring was constructed. Initially the protection
Cell migration inhibitors ideally could materialize the antimeta-
static treatment as they could suppress cell motility and even keep tu-
mors static specifically by interfering with cellular migration ma-
chinery. The last two decades have seen many migration inhibitors,
either isolated from natural sources or synthetized in laboratories.
These inhibitors include natural products such as (+)-migrastatin,4
stylissamide X,5 (+)-fusarisetin A,6 withaferin A,7 prodigiosin8 and
migracins A and B,9 and synthetic compounds like thiazole derivatives
as a fascin inhibitor10 and isoxazolines as an integrin antagonist and so
⁎ Corresponding authors.
E-mail addresses: yelong@wust.edu.cn (L. Ye), xinghualiao@wust.edu.cn (X.-H. Liao).
1 These two authors contribute equally.
https://doi.org/10.1016/j.bmcl.2019.126914
Received 1 October 2019; Received in revised form 5 December 2019; Accepted 16 December 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MianYang,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126914

M. Yang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
O
OEt
O
O
O
O
NH
OEt
S
S
N
S
N
N
d
c
a
b
BocHN
H2N
BocHN
S
O
N
H2N
N
3
2
1
4
5a/6a/7a, R1=4-H
5b/6b/7b,R1=4-CH3
5c/6c/7c, R1=4-CH3O
5d/6d/7d, R1=4-t-Bu
5e/6e/7e, R1=4-F
5f/6f/7f, R1=4-Cl
5g/6g/7g, R1=4-Br
5h/6h/7h, R1=4-I
5i/6i/7i, R1=3-Br
O
OEt
NH
O
AWD*I NH2
NH
O
OH
NH
N
N
N
e
f
O
O
O
S
S
S
N
H
N
H
N
N
R1
R1
N
N
R1
H
6
5j/6j/7j, R1=2-Br
5
7
Scheme 1. Synthesis of Substituted Benzamidothiazolylpyrazole-capped AWD*I-NH2. Reagents and conditions: (a) (Boc)2O, DMAP(cat.), TEA, THF, 70 °C, 100% (b)
Diethyl oxalate, LHDMS, THF, −78 °C, 84% (c) i) hydrazine hydrate, EtOH, 90 °C; ii) TFA, DCM, r.t., 73% (d) (R1)PhCO2H, CDI, DMF, 85 °C, 75–85% (f) i) NaOH
(1 M, a.q.), THF, r.t.; ii) HCl (1 M, a.q.), 70–80% (g) AWD*I-NH2, HATU, DIEPA, DMF, 0 °C-r.t. 48 h, 40–50%
reaction suffered a low yield when just Boc anhydride and a base were
mixed with the amine 1 in a solvent. After several trials with reaction
conditions, we found that addition of a catalytic amount of DMAP and
refluxing the reaction mixture could push the protection reaction go
complete, giving the N-Boc 2-amino thiazole derivative 2 in a quanti-
tative yield. Then the 5-acyl group of 2 was reacted with diethyl oxalate
via a Claisen condensation to form diketone 3 in a good yield. The
diketone then was condensed with hydrazine hydrate to generate a
pyrazole intermediate, which was subjected to solvent removal and TFA
treatment to cleave the Boc group attached to the 2-amino group on the
thiazole ring, resulting in 4. The amino group of 4 was then reacted
with a series of substituted benzoic acids respectively to yield sub-
stituted benzamidothiazolylpyrazoles 5. Saponification of the ester
group of 5 and subsequent acidification of the reaction mixture gave the
carboxylic acids 6 in the form of off-white precipitates. Each of these
acids then was reacted with the amino group of the alanine in the
peptide AWD*I-NH2 via a HATU-mediated amidation in solution phase,
delivering the final products 7 in the yield of 40–50%, which is a sig-
nificant increase in comparison to the very low yield of the solid-phase
coupling with resin-bound AWD*I-NH2.19 Initially the peptide even
failed to react with acids 6 under the same condition as the one for the
DIC/HOBt-mediated synthesis of 7a using the resin-bound peptide. The
initial failure might be because the peptide folded with more freedom
after it was cleaved off from the resin beads and its low energy con-
formation was able to form, resulting in a less accessible amino group of
the alanine in the molecular form of the peptide versus the resin-bound
peptide. After several rounds of experiments with different acyl-acti-
vating agents, it turns out that HATU could successfully mediate the
formation of the peptide-bisazole conjugate (7). Now that the pepti-
domimetics do not have to be made with the resin-bound peptide as
before, the production of these compounds is no longer restricted by the
loading limit of the resin and it could be carried out at any desired
scale. Besides the late-stage diversification of the biszaole capping
agents 6 and the solution-phase reaction between the capping agent and
the tetrapeptide, the above six-step synthesis of the peptidomimetics
also has several other advantages over our original on-bead synthesis of
the original hit (7a).19 One of the advantages is the improvement of
overall yield of the peptidomimetics due to the convergent synthetic
route in which 6 and the peptide were prepared separately and coupled
together at a final step. And the amount of acids 6 was much reduced
for the final coupling reaction whereas they had to be excessive when
reacting with the resin-bound peptide. Furthermore, the cryogenic
Claisen condensation needs to be executed only once in the synthesis of
the whole set of the analogs, whereas it would be repeated many times
if various 2-benzamidothiazoles were made first and then the dry ice-
chilled reaction would be run each time to form the pyrazole ring for
every different analog. In all words, the synthesis of the peptidomi-
metics was much streamlined in comparison to the previous linear
route.
To evaluate the effect of the peptidomimetics 7 on cancer cell mi-
gration, we started with a transwell assay using a highly metastatic cell
line, the breast cancer cells MDA-MB-231 as a test subject in the pre-
sence of these compounds and lipase sPLA2-IIA as a chemotaxi agent.
Based upon the reduction of migrating cells in the presence of each of
compounds 7, we obtained the rates of inhibition on the lipase-directed
cell migration by the compounds (the magenta markers in Figure 1).20
As Figure 1 shows, the peptidomimetics 7a through 7i have various
inhibition rates, ranging from 10% to 42%. Among the active analogs
7e have an inhibition rate larger than the initial hit 7a, indicating that
the 4-fluorine substituent on the phenyl ring could enhance the in-
hibitory effect of the peptidomimetic structure. Considering the fact
that a fluorine atom is much more electronegative than hydrogen but
comparable in size with hydrogen, it seems that the inhibitory effect
Figure 1. Inhibition rates of substituted (2-benzamidothiazol-5-yl)pyrazole-
capped AWD*I(NH2) on the motility of breast cancer cells MDA-MB-231 in both
transwell and scratch assay. Cells were dosed with the DMSO solution of 7a
through 7j, respectively. The concentrations of the compounds are 2 μM in the
transwell assay and 20 μM in the scratch assay respectively.
2

M. Yang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
might benefit more from the stronger electron-withdrawing ability of
the fluorine substituent. On the other side, electron-donating groups
such methyl, methoxy and t-butyl reduce the migration inhibitory po-
tency as demonstrated by decreased inhibition rates of analog 7b
through 7d. Especially, as methoxy is the strongest electron-donating
among the three substituents, the inhibition rate of the methoxy-sub-
stituted analog (7c) reaches the lowest within the three analogs. Among
the halogenated analogs 7e through 7h, the inhibition rate decreases
continuously as the periodic number of the halogen substituent in-
creases, implying that the smaller and more electron-withdrawing ha-
logen substituent could enhance the inhibitory effect on cell migration,
whereas the larger and less electron-withdrawing one would undermine
the effect. Among the three brominated analogs (7g, 7i and 7j), 7i has
the largest inhibition rate, meaning that the bromine substitution at the
phenyl meta position is beneficial to the inhibition potency. The better
activity of the meta-bromo substituted analog might be due to bromine’s
stronger electron-withdrawing force at the phenyl meta position than at
para or ortho positions.21 Considering the above structure–activity re-
lationships, it seems that the electron-withdrawing capacity of the
substituent could be the potency-increasing factor for the peptidomi-
metics with the same type of substituents. One of the consequences with
a stronger electron-withdrawing substituent on the phenyl ring is the
increased acidity of the adjacent benzamide group, making it a better
hydrogen donor, which might render a stronger binding with a target
protein and possibly a higher level of antimigration activity. This kind
of correlation between the acidity of an amide group and the anti-
migration potency of the whole molecule was also observed among the
isoxazoline-based cell migration inhibitors.11 In addition, the results of
the inhibition rates also indicate that there might be some other factors
that could influence the potency of the migration inhibitors because the
chlorinated analog 7f is less active than the methylated one 7b even
through a chlorine atom is more electron-withdrawing than a methyl
group and comparable in size with the group. One plausible reason is
that a chlorine atom and a methyl group differ not only in electron-
withdrawing capacity but also in some other electrosteric aspects such
as static field and shape etc. More in-depth study is needed to under-
stand the structure–activity relationship of these compounds.
Figure 2. The cell viability of MDA-MB-231 cells in the presence of the pepti-
domimetics 7. It was measured with CCK8-kit after cells were dosed with the
DMSO solution of the 7a through 7j, respectively, or neat DMSO as a reference
(V.C.) and then incubated for 48 h.
cell viability assay to check if there is a correlation between the two
effects on cells. By comparing the amount of a metabolite generated by
life dosed cells with the amount of the same metabolite by life undosed
cells, we obtained the cell viability in the presence of compounds 7
(Figure 2).23 The figure indicates that cells treated with all of the
compounds at 1 μM concentration had no significant loss in viability,
meaning these compounds affect little toxicity to the breast cancer cells
at the concentration. The viability decreased as the concentration of the
compounds was increased up to 10 μM. However, most analogs except
7j still imposed no significant toxicity since their viability values re-
main around 90%. And the most potent migration inhibitors (7e) did
not affect any viability loss at both concentrations. The other analogs
such as 7a and 7d did not have their cell viability significantly reduced
either. The most toxic analog is 7j, which reduced cell viability by al-
most 30% at 10 μM, is the least potent inhibitors. Therefore, the cy-
totoxicity of the compounds is not correlated with their migration in-
hibition capacity. Therefore, the migration inhibitory effect of these
compounds is least likely due to their cytotoxicity.
To confirm the antimigration effect of the peptidomimeitcs in a
different setting, we performed a scratch assay with the same line of
cancer cells without using lipase sPLA2-IIA to induce cell migration.
Based on the reduction in the area recovered by dosed cells in com-
parison to the undosed ones in the absence of the chemotaxi agent, we
acquired the rates of inhibition on cells’ spontaneous migration by the
compounds 7 (the blue markers in Figure 1).22 As the blue markers
show, all the peptidomimetic analogs have significant inhibition rates
(15–49%) against cell migration even though no exogenous chemotaxi
agent was added. These data suggest that the compounds could take
effect on the intrinsic cellular motility as well. Also the scratch in-
hibition rates of 7 show the same trend as the transwell inhibition rates
of these compounds. For example, the analog with the 4-methoxy
substituent remains the least active among the alkyl and alkoxy sub-
stituted analogs (7a through 7d). Also the fluorinated analog 7e still
has the strongest potency among all analogs. And the scratch inhibition
rate also decreases from 7e through 7h as the periodic number of the
halogen substituent increases. At last, the analog with the 3-bromo
substituent also has a larger inhibition rate than the other bromo ana-
logs in the scratch assay. However, there still are some differences be-
tween the two inhibition rates. One difference is that the scratch in-
hibition rates of the most analogs are larger than the transwell ones.
This might be due to the higher concentration of the compounds tested
in the scratch assay. The larger inhibition rates at the higher con-
centration of the most analogs imply that there might be a good cor-
relation between the migration inhibition and the concentration of the
peptidemimetics.
After the antimigration effect of peptidomimetics 7 were confirmed
in the above assays, we then checked if the effect was specific to the
breast cancer cells. Since compound 7e has the most potency and no
apparent cytotoxicity, we run a transwell migration assay with two
other metastatic cells, such as lung cancer cells A549 and cervical
cancer cells HELA using the same assay parameters as with the breast
cancer cells. In the assay the A549 and HELA cells were dosed with the
DMSO solution of the compound and cells in a control well were treated
with the same volume of neat DMSO solution as of the compound so-
lution. After all the cells were incubated as in the previous transwell
assay, the cells that migrated into the lower chamber were counted. The
percentage ratio of the cell counts of dosed migrating cells to that of the
migrating cells in the control well were calculated and shown as re-
lative migration rates in Figure 3. As shown in the figure, the relative
migration rates of both A549 and HELA cells were reduced to about
60% of the one of the vehicle control after the cells were dosed with
compound 7e, meaning the compound can significantly inhibit the
migration of both cancer cells by about 40%, which is a comparable
potency as in the assay with the breast cancer cells. Also, our previous
work indicates that 7a could stop cells U937 from migrating.19 There-
fore, as this type of the compound demonstrated the inhibitory effect on
migration of at least the four lines of cancer cells in total, it would serve
as a valuable lead structure for the development of antimetastatic
therapeutics against a broad spectrum of cancers.
To rule out the possibility that the antimigration activity of the
peptidomimetics 7 is attributable to their cytotoxicity, we performed a
As compound 7e demonstrated the strongest inhibitory effect on cell
migration in the previous assay, we chose this compound to study the
3

M. Yang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
aUndosed Cells
A549
HELA
ratio of the counts of dosed migrating cells to the count of the migrating
cells in the control well (Figure 4). As shown by the figure, the relative
migration rate of the cancer cells decreases continuously from about
100% to about 20% as the concentration increases from 0.0 μM to
50 μM. That is to say that the higher dose of the compound, the less
cells migrated and the stronger inhibitory effect of the compound.
Therefore, the migration inhibitory effect of the peptidomimetics is
constantly in action as long as the dose of the compound is not zero and
the inhibitory intensity is proportional to the dosage. Accurate mathe-
matic correlation equation will be established later in a more refined
experiment. Here the figure indicates that the IC50 of the migration
inhibitor is around 5.0 μM.
150
100
50
*
*
0
d
9
54
A
e
s
EL
o
A
d
H
n
U
Figure 3. The relative migration of two different cancer cells, A549 and HELA
in the presence of 7e, of which concentration is 5 μM. Neat DMSO in place of
the compound solution as a reference. aUndosed A549 or HELA cells, *:
p < 0.05 verse control.
To preliminarily investigate the mechanism underlying the anti-
migration effect of the peptidomimetics, we checked whether the level
of f-actin in MDA-MB-231 cells was changed after cells were dosed with
the compound 7e as it is a key molecular assembly for cell’s locomotive
protrusions such as lamellipodia during cell migration.24 In this assay f-
actin in the cells that were or were not treated with compound 7e was
visualized with red Alexa548-phalloidin as shown in Fig 5.25 The figure
shows that the red color in the 7e-dosed cells is much thinner than in
the vehicle control, meaning less f-actin formed in the cells dosed by the
migration inhibitor 7e. Therefore under the influence of the peptido-
mimetic compound the cells could not form locomotive protrusions due
to the lack of f-actin, resulting in the loss of cellular motility.
In summary, we achieved the efficient and scalable synthesis of the
substituted benzamidothiazolylpyrazole-capped AWD*I-NH2 via the
late-stage diversification and solution-phase coupling between the bi-
sazole capping agent and the peptide motif. Most of the analogs have
the antimigration activity as the initial hit does but their potencies vary
with the substitution on the terminal phenyl ring. In general, electron-
withdrawing substituent could enhance the potency. Also, the migra-
tion-inhibiting effect of this type of compound holds against multiple
cancer cell lines and most active peptidomimetics showed no apparent
cytoxicity, suggesting its good potential for the development of anti-
metastatic therapeutics. The migration inhibitory effect is partially
linked to the reduction in f-actin. More effort involving target identi-
fication is taken to investigate the detailed molecular mechanism of the
antimigration effect.
Figure 4. The result of the dose–response experiment with 7e. The cells were
dosed with the compound at different concentrations, respectively. Cellular
relative migration decreases as the concentration of 7e increases. Statistical
analysis of different concentration cell migration levels. *: p
control, **: p < 0.01 verse control.
< 0.05 verse
DAPI
f-actin
merge
Acknowledgments
Thanks are given to Overseas Scholar Starting Fund by China
Education Minister (Batch 48), Natural Science Fund of Hubei Province
(2015CFB490) and Open Fund of China Education Minister Key
Laboratory for Medical Polymers at Wuhan University (20160101).
Special thanks are given to Professor Xian-Zheng Zhang and Professor
Si-Xue Cheng at Wuhan University for their generous technical sug-
gestions and financial support. The authors also would like to thank
Professor Yi Liu and Qiang-hui Zhou at Wuhan University for providing
assistance in high resolution mass spectrometric analysis.
VC
7e
Appendix A. Supplementary data
Figure 5. The microscopic images of localization of DAPI (blue) on cell nucleus
and phalloidin on f-actin (red) in MDA-MB-231 cells treated with compound 7e
or DMSO vehicle control (VC). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.126914.
References
dose–response correlation to see how the effect varies with the con-
centration of the inhibitor. In this study we performed a transwell assay
still with the breast cancer cells MDA-MB-231 and dosed them with a
DMSO solution of the compound at a series of different concentrations.
Again in a control well was just added the same volume of neat DMSO
as of the compound solution. After incubation as before, only the cells
that moved into the lower chamber were counted. Relative migration
rates for all concentrations were obtained by calculating the percent
1. Chambers AF, Groom AC, MacDonald IC. Nature Rev. Cancer. 2002;2:563.
2. Fidler IJ. Nature Rev. Cancer. 2003;3:453.
3. Hanahan D, Weinberg RA. Cell. 2011;144:646.
4. Nakae K, Yoshimoto Y, Sawa T, et al. J. Antibiot. 2000;53:1130.
5. Arai M, Yamano Y, Fujita M, Setiawan A, Kobayashi M. Bioorg. Med. Chem. Lett.
1818;2012:22.
6. Xu J, Caro-Diaz EJE, Lacoske MH. Chem Sci. 2012;3:3378.
7. Lee J, Hahm ER, Singh SV. Carcinogenesis. 1991;2010:31.
8. Zhang J, Shen Y, Liu J, Wei D. Biochem. Pharmacol. 2005;69:407.
9. Arai Y, Iinuma H, Ikeda Y, et al. Antibio. 2013;66:225.
4

M. Yang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
10. (a) Zheng S, Zhong Q, Jiang Q, et al. ACS Med. Chem. Lett. 2013;4:191
(b) Zheng S, Zhong Q, Xi Y, et al. J. Med. Chem. 2014;57:6653.
22. Cells were seeded into 12-well plates and grown to about 80% confluence on the
bottom of each well. Then micropipette tips were used to make a cell-free scratch on
the bottom. Washed off suspended cells with PBS buffer followed by addition of a
regular serum and the DMSO solution of one of the compounds 7 or neat DMSO in a
control well. Then all the cells were incubated in 5% CO2 at 37°C for 48 h. The
scratch areas were covered again by migrating cells during the incubation. The
percentage ratio of the difference between the area newly covered by the dosed cells
and the one by the reference cells to the area newly covered by the reference cells
was calculated for each compound as the scratch inhibition rate of the compound
(the blue markers in Figure 1).
11. Pitts William J, Wityak John, Smallheer Joanne M. J. Med. Chem. 2000;43:27.
12. Elsayed HE, Akl MR, Ebrahim HY, et al. Chem. Biol. Drug Design. 2015;85:231.
13. Carriero MV, Ciliberto G, Bifulco K, Pessi A. PCT Int. Appl.
2017;WO2017178333:A1–20171019.
14. Skruber K, Chaplin KJ, Phanstiel O. J. Med. Chem. 2017;60:8606.
15. Nandakumar N, Muthuraman S, Gopinath P, Nithya P, Gopas J, Kumar RS. Euro. J.
Med. Chem. 2017;125:1076.
16. Njardarson JT, Gaul C, Shan D, Huang XY, Danishefsky SJ. Discovery of potent cell
migration inhibitors through total synthesis: lessons from structure-activity studies of
(+)-migrastatin. J. Am. Chem. Soc. 2004;126:1038.
23. MDA-MB-231 cells were placed in a 96-well plate at the density of 1.0×105 and then
treated with the DMSO solution of one of the compounds 7 or neat DMSO in a
control. After 48 hours of incubation, cells were dosed with compound WST-8 from a
CCK-8 kit and incubated for another 1 hour and then the optical density (OD) value
of the cellular metabolite of WST-8, formazan was recorded using a microplate
reader. The percent ratio of the OD value for the dosed cells to that for the undosed
cells is calculated and termed as relative cell viability (Figure 2).
17. Gaul C, Njardarson JT, Shan D, et al. J. Am. Chem. Soc. 2004;126:11326.
18. Shan D, Chen L, Njardarson JT, et al. Proc. Natl. Acad. Sci. USA. 2005;102:3772.
19. Ye L, Dickerson T, Kaur H, et al. Bioorg. Med. Chem. Lett. 2013;23:340.
20. Cells were first seeded into the upper Boyden chamber at a density of 1.0×105 in a
media free of serum. To the lower chamber were added 10% FBS and 2.5 nM sPLA2-
IIA. Then the cells were dosed with the DMSO solution of compound 7 or neat DMSO
as a reference in a control well. After the cells were incubated in 5% CO2 at 37°C for
12 hours, cells which migrated into the lower chamber were counted under a mi-
croscope. The percentage ratio of the difference between the cell count of the re-
ference and the one of the dosed cells to the cell count of the reference was calculated
as the transwell inhibition rate of an individual compound (the magenta markers in
Figure 1).
24. Lauffenburger DA, Horwitz AF. Cell. 1996;84:359.
25. Cells were grown on collagen-1 coated glass cover slips first and then cultured for 16
h in a serum-free medium containing the DMSO solution of compound 7e at 5 μM or a
neat DMSO solution in a vehicle control. The cells were then fixed with PFA, per-
meabilized with Triton X-100 and BSA in PBS. The f-actin in the cells was specifically
stained by red Alexa548-phalloidin and nuclei of the cells by blue DAPI, respectively.
After the staining, the cells were put under a laser scanning confocal microscope for
visualization and the observed images of the cells are shown in Figure 5.
21. Ritche CD, Sager WF. Prog. Phys. Org. Chem. 1964;2:323.
5