Discovery of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as new potent and selective human sirtuin 2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.04.010

Human sirtuin 2 (SIRT2) plays pivotal roles in multiple biological processes such as cell cycle regulation, autophagy, immune and inflammatory responses. Dysregulation of SIRT2 was considered as a main aspect contributing to several human diseases, including cancer. Development of new potent and selective SIRT2 inhibitors is currently desirable, which may provide a new strategy for treatment of related diseases. Herein, a structure-based optimization approach led to new 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as SIRT2 inhibitors. SAR analyses with new synthesized derivatives revealed a number of new potent SIRT2 inhibitors, among which 28e is the most potent inhibitor with an IC₅₀ value of 42?nM. The selectivity analyses found that 28e has a very good selectivity to SIRT2 over SIRT1 and SIRT3. In cellular assays, 28e showed a potent ability to inhibit human breast cancer cell line MCF-7 and increase the acetylation of α-tubulin in a dose-dependent manner. This study will aid further efforts to develop highly potent and selective SIRT2 inhibitors for the treatment of cancer and other related diseases.

Full text of DOI:10.1016/j.ejmech.2017.04.010

Accepted Manuscript
Discovery of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as
new potent and selective human sirtuin 2 inhibitors
Lingling Yang, Xiaobo Ma, Chen Yuan, Yanying He, Ling Li, Sha Fang, Wei Xia, Tao
He, Shan Qian, Zhihong Xu, Guobo Li, Zhouyu Wang
PII:
S0223-5234(17)30265-9
10.1016/j.ejmech.2017.04.010
EJMECH 9357
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2016
Revised Date: 1 April 2017
Accepted Date: 6 April 2017
Please cite this article as: L. Yang, X. Ma, C. Yuan, Y. He, L. Li, S. Fang, W. Xia, T. He, S. Qian, Z. Xu,
G. Li, Z. Wang, Discovery of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as new
potent and selective human sirtuin 2 inhibitors, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.04.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives
as new potent and selective human Sirtuin 2 inhibitors
Lingling Yang†*, Xiaobo Ma‡, Chen Yuan†, Yanying He†, Ling Li†, Sha Fang†, Wei
Xia†, Tao He‡, Shan Qian, Zhihong Xu‡, Guobo Li§, Zhouyu Wang‡*
†College of Food and Bioengineering, Xihua University, Sichuan 610039, China
‡College of Science, Xihua University, Sichuan 610039, China
§Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of
Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
*To whom correspondence should be addressed. Tel.: +86-28-7725898;
Fax:+86-28-87725898;E-mail: yangll0808@sina.com, zhouyuwang77@gmail.com
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Abstract
Human sirtuin 2 (SIRT2) plays pivotal roles in multiple biological processes such as
cell cycle regulation, autophagy, immune and inflammatory responses. Dysregulation
of SIRT2 was considered as a main aspect contributing to several human diseases,
including cancer. Development of new potent and selective SIRT2 inhibitors is
currently desirable, which may provide a new strategy for treatment of related
diseases. Herein,
a
structure-based optimization approach led to new
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as SIRT2
inhibitors. SAR analyses with new synthesized derivatives revealed a number of new
potent SIRT2 inhibitors, among which 28e is the most potent inhibitor with an IC50
value of 42 nM. The selectivity analyses found that 28e has a very good selectivity to
SIRT2 over SIRT1 and SIRT3. In cellular assays, 28e showed a potent ability to
inhibit human breast cancer cell line MCF-7 and increase the acetylation of α-tubulin
in a dose-dependent manner. This study will aid further efforts to develop highly
potent and selective SIRT2 inhibitors for the treatment of cancer and other related
diseases.
Keywords: Sirtuins; SIRT2; Epigenetics; Cancer; Structure-based optimization
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1. Introduction
Sirtuins (SIRTs) were initially recognized as NAD+-dependent protein deacetylases
that remove acetyl groups from ɛ-N-acetyl-lysine amino groups on histones and
non-histone substrates, e.g. α-tubulin[1,2]. Recently, sirtuins were found to catalyze
further post-translational modifications such as desuccinylation[3], demalonylation[3],
demyristoylation[4], and ADP-ribosylation[5,6]. By modifying various substrates,
sirtuins are involved in multiple crucial physiological processes, including the
regulation of metabolism, transcription, and DNA damage repair[1,2,7-10]. In human
the sirtuin family comprises seven proteins (SIRT1-SIRT7), which vary in their
catalytic activity and subcellular localization. The human sirtuin 2 (SIRT2), located in
both cytoplasm and nucleus, mainly catalyzes deacetylation or demyristoylation for a
variety of protein substrates, including histones H3[11] and H4[12], and non-histone
proteins α-tubulin[13], p53[14], p65[15], Foxo1[16], and Foxo3a[17]. SIRT2 thus has
been shown to play important roles in cell cycle regulation, autophagy, peripheral
myelination, immune and inflammatory response[1,7,8].
Overexpression or dysregulation of SIRT2 was considered as a main aspect
contributing to several human diseases, including cancer[1,8]. For example, Chen et
al recently found that SIRT2 is overexpressed in hepatocellular carcinoma, and
mediates epithelial to mesenchymal transition (EMT) by regulating the deacetylation
of protein kinase B and subsequently influencing the glycogen synthase
kinase-3β/β-catenin signaling pathway[18]. Many other studies revealed that SIRT2 is
up-regulated in several human cancers such as breast cancer and pancreatic
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adenocarcinomas, and SIRT2 down-regulation or inhibition leads to cancer cell
apoptosis or growth suppression[19,20]. Although SIRT2 was also observed to have
cancer-suppressing activity in some types of cancer[21], the development of SIRT2
inhibitors, especially potent and selective inhibitors, would be an impetus to probe
associated molecular mechanism and to develop therapeutic drugs for the treatment of
selected cancer types.
To date, various structurally diverse types of SIRT2 inhibitors have been reported
(1-11, Figure 1)[22-28], most of which showed only moderate potency or non-specific
inhibition to SIRT2. Notably, Jung and coworkers recently discovered aminothiazoles
as selective SIRT2 inhibitors termed SirReals (9, Figure 1), which can induce/bind to
a new selective binding pocket (surrounded by almost all the hydrophobic residues
such as Phe96, Phe190, Ile169, Leu134, Ile232, Phe119, Leu138, Tyr139, Pro140, and
Phe143) as revealed by crystallographic analyses[27,29,30].
With the aim of identifying new selective SIRT2 inhibitors, we initially carried
out structure-based design by LEADOPT, an automatic lead optimization tool
developed recently by us, which enables generation of new derivatives for the given
lead compound and its core scaffold according to the target information (details can
be found in our previous work[31]). The Sirt2:SirReal2 (9, Figure 1) complex
structure (PDB ID: 4RMG)[29] was selected as the template, and the
2-((4,6-dimethylpyrimidin-2-yl)thio)acetamide motif (shown in red, Figure 2) was
defined as the core scaffold. The LEADOPT computations led to the generation of
1341 molecules, among which 422 molecules have a ligand efficiency (LE) score
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larger than 0.38 (an empirical LE score) and an ADMET score larger than 9 (an
ADMET score of SirReal2). From them, we selected compounds I and II to
synthesize (Figure 2) because both have never been reported and can be readily
synthesized (Scheme 1); the moieties of I and II (3-phenoxyphenyl and
3-((thiophen-2-ylmethyl)amino)phenyl, respectively) introduced by LEADOPT
appear positioned to form hydrophobic interactions with Phe96, Phe190, Ile169,
Leu134, Ile232, and Phe119. The inhibition assays showed that I and II displayed
92±3% and 93±1% inhibition against SIRT2 at 50 µM, and 49±5% and 60±2%
inhibition at 5 µM (Table 1), respectively. These preliminary results indicated that I
and II could be as potential hit compounds for further structural optimization to
obtain potent and selective SIRT2 inhibitors. We herein describe structure-activity
relationship (SAR) of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide (the
core chemical scaffold of compounds I and II) derivatives with SIRT2, and
anti-cancer activity for the most potent compound.
<Figure 1 here>
<Figure 2 here>
2. Results and Discussion
2.1 Synthesis
derivatives.
of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide
We synthesized a series of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenyl acetamide
derivatives to study the SAR with SIRT2 by the synthetic routes outlined in Schemes
1-3. A general scheme for the synthesis of compounds 14a-j and 15a-b is shown in
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Scheme 1. Reactions of commercially available 3-/4-substituted anilines (12a-b, 12d,
and 12g-h) or synthesized 3-/4-substituted anilines (12c, 12e-fand 12i-j, see Scheme
S1) with 2-bromoacetic acid in the presence of 1-hydroxybenzotriazole (HOBT),
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI), and
N,N-diisopropylethylamine (DIPEA), produced the condensation products 13a-j. The
resulting key intermediates 13a-j were subsequently reacted with
4,6-dimethylpyrimidine-2-thiolin the presence of t-BuoK at room temperature to give
the desired compounds 14a-j in 56%-88% yields. Further oxidization of compound
14b by 1.0 equiv or 3.0 equiv m-CPBA led to the white solid compounds 15a-b,
respectively.
The synthesis of compounds 19a-f and 24a-b, which contain a CH2-NH or NH-CH2
linker
substituted
at
the
benzene
ring
of
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenyl acetamide, are depicted in Scheme 2.
The compounds 19a-f containing a CH2-NH linker, were obtained by starting from the
condensation reaction between p/m-phenylenediamine with 2-bromoacetic acid,
selectively. Then, the nucleophilic substitution and reductive amination reaction were
performed in sequence to produce the final products 19a-f in high yields. For the
NH-CH2 linker compounds 21a-b, a two-step reductive amination reaction between
3/4-nitrobenzaldehyde with aniline resulted in the intermediates 21a-b. Then the
target compounds 24a-b were synthesized from 21a-b using a method similar to that
for 14a-j in moderate yields.
Scheme 3 presents the synthetic routes for compounds 28a-k, 32 and 35, which
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contain an O-CH2 or CH2-O linker. For the synthesis of O-CH2 linker compounds
28a-k, the target compound 28e was described as an example. First, commercially
available1-(bromomethyl)-3-nitrobenzene
(25)
was
reacted
with
N-(4-hydroxyphenyl)acetamide by a base-promoted nucleophilic substitution
followed by a deacetylation reaction in the presence of thionyl chloride and methanol.
Next, the free amino group was reacted with thiophene-2-carbonyl chloride in a
standard condensation of chloride and amine to yield the key intermediates 26e,
which was reduced to give the compound 27e in a high yield. Finally, 28e was
obtained using a two-step procedure involving condensation reaction and final
nucleophilic substitutionas described above. For the CH2-O linker contained
compounds 32 and 35, intermediates 3-(benzyloxy)aniline (31) and
4-(benzyloxy)aniline (34) were used to react with 2-bromoacetic acid and
4,6-dimethylpyrimidine-2-thiol by general procedure 1 and general procedure 2 (see
experimental section) in turn, respectively. Note, the key intermediates 31 and 34
were synthesized through different synthesis methods, which were described in the
experimental section.
2.2 SAR studies with SIRT2.
In
order
to
explore
SAR
of
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives with SIRT2, we
firstly synthesized compounds 14a-j (Table 1) containing oxydibenzene,
diphenylamine, diphenylmethane, benzophenone and diphenylmethanol at 3- or
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4-position of the phenyl of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide
scaffold (Table 1). Compared with the hit compound I (14a), which has an
oxydibenzene motif at the 3-position, compound 14b, containing an oxydibenzene
motif at 4-position, showed slightly lower potency to inhibit SIRT2 (78±3% and 42±4%
inhibition at 50µM and 5 µM, respectively). A further comparison of the compound
pairs [14c vs 14d], [14e vs 14f], [14g vs 14h], and [14i vs 14j], which contain
diphenylamine, diphenylmethane, benzophenone and diphenylmethanol at 3- or
4-position, respectively, revealed that 3-substituted derivatives are likely to have more
potent inhibition against SIRT2 than 4-substituted derivatives (Table 1). Notably, 14e,
containing a diphenylmethane motif at 3-position, displayed promising potency with
96±3% and 73±4 % SIRT2 inhibition at 50µM and 5 µM, respectively, which is more
potent than the known SIRT2 inhibitor Suramin (Table 1). Furthermore, we
synthesized compounds 15a and 15b, which are the oxidized form on thio group of
14b. Comparing with 14b, the activities of compounds 15a and 15b against SIRT2
significantly decreased (Table 1), indicating that the oxidized form of the
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide
may
destruct
the
intermolecular hydrogen bond between dimethylpyrimidine and amide, which is an
very important feature of the inhibition mode as observed in previous crystallographic
analyses[29,30].
<Table 1 here>
According to the hit compound II (19a), we synthesized the target compounds
19b-f (CH2-NH linker), 24a-b (NH-CH2 linker), 32 (CH2-O linker), 35(CH2-O linker),
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and
28a-b
(O-CH2
linker)
(Table
1),
which
contain
((thiophen-2-ylmethyl)amino)phenyl, (benzyloxy)phenyl, ((phenylamino)methyl)
phenyl, (benzylamino)phenyl, ((pyridin-4-ylmethyl)amino)phenyl, ((furan-2-yl
methyl)amino)phenyl and (phenoxymethyl)phenyl at 3- or 4-position. We observed
that 19b, bearing a ((thiophen-2-ylmethyl)amino)phenyl motif at 4-position, show
lower SIRT2 inhibition than the hit compound II (19a), which has a
((thiophen-2-ylmethyl)amino)phenyl motif at 3-position. Consistent with the SAR
described above, compounds 19c, 24a, 32, and 28a, which have (benzyloxy)phenyl,
((phenylamino)methyl)phenyl, (benzylamino)phenyl, and (phenoxymethyl)phenyl at
3-position, respectively, manifested more potent inhibition against SIRT2 than
compounds 19d, 24b, 36, and 28b with corresponding substituents at 4-position.
These results indicated that substituents at 3-position of the phenyl of
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide may be beneficial to fit with
the induced selective SIRT2 binding pocket. Notiably, among these tested compounds,
28a with a (phenoxymethyl)phenyl moiety at 3-position showed the very potent
inhibition towards SIRT2 (71±3% and 96±3% inhibition at 5µM and 50µM,
respectively). Molecular docking analyses indicated that 28a appears positioned to
make good hydrophobic interactions with Phe119, Ile232, Phe131, Leu134, Leu138,
and Phe96 (Figure 3a); compared with compound I, the (phenoxymethyl)phenyl
moiety of 28a likely binds adjacent to the hydrophilic region (such as Val233 and
His187, Figure 3a), suggesting that modification of the A moiety or introduction of
substituents at 3- or 4-position of the A moiety of 28a (Table 2) would probably
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improve SIRT2 inhibitory activity.
<Figure 3 here>
<Table 2 here>
We further synthesized compounds 28c-k, which contain various substituents on
or variants of the phenyl moiety (the A moiety, Table 2) of 28a, using the routes given
in Scheme 3. Inhibition assays revealed that substituents at the A moiety of 28a
showed comparable or improved SIRT2 inhibitory activities to 28a (Table 2).
Compound 28e, which contains a thiophene-2-carboxamide moiety as the A moiety of
28a, displayed very potent inhibition to SIRT2 (98 % inhibition at 5µM). We also
observed that compounds 28h, 28i, 28j, and 28k, which have 1-naphthalene,
2-naphthalene, 8-quinoline, or 5-1H-indazole as the A moiety, showed moderate
SIRT2 inhibitory activity but better than Surinam. Through molecular docking
anlayses, we observed that, comparing with 28a, the thiophene-2-carboxamide moiety
of 28e is likely to have additional hydrogen-bonding interactions with Val233 and π-π
stacking interactions with His187, which may explain why 28e has more potent
inhibition activity than 28a. Together, these SAR studies led to a series of new
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives as SIRT2
inhibitors, and especially the most potent compound 28e.
2.3 The selectivity of compound 28e.
For compound 28e, we further determined the IC50 value against SIRT2. As
shown in Figure 4a, 28e inhibit SIRT2 via a dose-dependent manner with an IC50
value of 42nM, which is more potent than Suramin (with an IC50 value of 14 µM,
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Figure 4b). We further tested compound 28e against SIRT1 and SIRT3, which are
close analogues to SIRT2, with the aim of testing the selectivity of 28e. We observed
that 28e did not show obvious inhibition to SIRT1 and SIRT3 even at the highest
concentration of 300 µM (Figure 4c and 4e); for comparison, the SIRT1 inhibitor
EX-527 and SIRT3 inhibitor SRT1720 displayed potent inhibition with IC50 values of
0.13 µM and 0.85 µM, respectively, under the same assay conditions (Figure 4d and
4f). These results indicated that 28e has good selectivity to SIRT2. It is possible that
28e as well as other derivatives (e.g. 28a) probably inhibit SIRT2 via binding to the
selective binding pocket[29,30].
<Figure 4 here>
2.4 Cellular Activities of 28e.
We tested whether compound 28e inhibit human breast cancer line MCF-7, which
was reported to be associated with the SIRT2 activity[26]. MCF-7 cells were treated
with 28e in four different concentrations (1.8 µM, 5.5 µM, 16.6 µM, and 50 µM) for
120 hours. We observed that compound 28e displayed a dose-dependent inhibition to
MCF-7 with more than 50% inhibition at 50 µM (Figure 5a). For comparison, we
tested compound 28b with a (phenoxymethyl)phenyl moiety at 4-position (Table 2),
which shows much lower inhibitory activity against SIRT2 than 28e; 28b did not
show obvious inhibition against MCF-7 at 50 µM (Figure 5a). Meanwhile, we tested
the inhibitory acitvities of compounds 28e and 28b against human normal liver cell
line HL-7702, which was used for ruling out the activities due cytotoxicity, We
observed that compounds 28e and 28b had no activity against HL-7702 at 100µM
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(with 3% and 6% inhibition @100µM, respectivly), suggesting that 28e and 28b are
not cytotoxic compounds. We then examined the effect of 28e on the acetylation of
α-tubulin, one of the key protein substrate of SIRT2. The results from western blotting
analysis showed that the acetylation of α-tubulinin MCF-7 cells was increased after
28e treatment for 6h (Figure 5b). These results indicated that 28e may inhibit MCF-7
by suppressing the SIRT2 activity and related signaling pathways.
<Figure 5 here>
3. Conclusion.
A series of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-phenylacetamide derivatives
were synthesized according to the hit compounds I and II obtained by LEADOPT.
The SAR analyses on these synthesized derivatives led to the identification of a
number of new potent SIRT2 inhibitors, e.g. 28e (with an IC50 value of 42 nM). The
selectivity analyses revealed 28e has a good selectivity to SIRT2 over SIRT1 and
SIRT3. 28e displayed inhibition to MCF-7 in a dose-dependent manner, which
showed a potent ability to increase the acetylation of α-tubulin as observed in western
blotting analyses. This study will aid further efforts to discover highly potent and
selective SIRT2 inhibitors and to develop SIRT2 inhibitors as a newstrategy for the
treatment of cancer as well as other associated diseases.
4. Experimental
Chemistry Methods
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All chemicals were obtained from commercial purchase and used as supplied without
further purification. Reactions were monitored by thin layer chromatography (TLC)
1
13
on Merck silica gel 60 F-254 thin layer plates. H NMR and C NMR spectra were
recorded on a Bruker AV-400 spectrometer at 400 MHz and 100 MHz, respectively.
Chemical shifts are given in ppm (d) relative to tetramethylsilane as an internal
standard or the NMR solvent. Low-resolution and high-resolution mass spectral (MS)
data were acquired on an Agilent 1100 series LC-MS instrument with UV detection at
254 nm in electrospray ionization (ESI) mode. Melting points were measured on an
electrothermal melting point apparatus without correction. All the target compounds
were purified to >95% purity, as determined by high-performance liquid
chromatography (HPLC). HPLC analysis was performed on a Waters 2695 HPLC
system equipped with a Kromasil C18 column (4.6 mm × 250 mm, 5 um).
General procedure 1: EDCI-mediated amide formation
A mixture of 2-bromoacetic acid(1.0 equiv), 1-hydroxybenzotriazole (HOBT,1.0
equiv), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.0
equiv) and N,N-diisopropylethylamine (DIPEA, 1.5 equiv) in dichloromethane (DCM)
was stirred at ambient temperature for 0.5 h. To the solution of the mixture, different
amines (1.0 equiv) were added, and the reaction mixture was continued to stir 10-12 h
at room temperature[32,33]. Upon completion of the reaction as determined by TLC,
The mixture was concentrated and partitioned between water and ethyl acetate. The
organic layer was dried over magnesium sulfate anhydrous, filtered and concentrated
in vacuo. The products were purified bycolumn chromatography with appropriate
eluents.
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General procedure 2: t-BuOK-catalyzed nucleophilic substitution
A solution of the 2-bromoacetamides (1.0 equiv), obtained via general procedure1,
4,6-dimethylpyrimidine-2-thiol (1.2 equiv) and t-BuOK (2.0 equiv) in DMF (10
mL/mmol) was stirred at ambient temperature for 5-12h. When TLC indicated that the
reaction was finished, the reaction mixture was poured into a large amount of water
(20ml/1ml DMF) and the crude mixture was extracted with ethyl acetate (3×). Then,
the combined organic layers were washed with brine, dried, and concentrated. The
residue was purified by column chromatography to give the desired target compounds
or key intermediates.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(3-(phenylamino)phenyl)acetamide (14c)
A mixture of 3-nitroaniline (1.2 equiv), phenylboronic acid (1.0 equiv), DBU (2.0
equiv), NiCl2.6H2O (0.2 equiv), and 2,2'-(C5H4N)2 (equiv) in MeCN (10ml/mmol)
was stirred for 28h at room temperature, Upon completion of the reaction as
determined by TLC, The mixture was concentrated and partitioned between water and
ethyl acetate. The organic layer was dried over magnesium sulfate anhydrous, filtered
and concentrated in vacuo. The solid was purified by column chromatography to give
the key intermediate 12cb. Though the Fe-mediated reduction reaction, the
N1-phenylbenzene-1,3-diamine (12c) was synthesized (Scheme S1). Using 12c, the
title compound 14c was obtained by general procedure 1 and general procedure 2 in
1
turn, 49% for two steps, 97.1% HPLC purity. mp: > 300°C; H NMR (400 MHz,
DMSO): δ 10.12 (s, 1H), 8.18 (s, 1H), 7.46 (s, 1H), 7.24 (t, J=8.0 Hz, 2H), 7.14 (t,
J=8.0 Hz, 1H), 7.08 (d, J=7.6 Hz, 2H), 7.01 (d, J=8.8 Hz, 1H), 6.98 (s, 1H), 6.83 (t,
J=7.2 Hz, 1H), 6.74 (dd, J=1.6 Hz, J=5.6 Hz, 1H), 4.02 (s, 2H),2.35 (s, 6H) ppm. 13C
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NMR (100 MHz, DMSO): δ 169.8, 167.4, 166.9, 140.3, 140.0, 139.5, 129.0, 128.8,
128.6, 125.5, 124.1, 119.7, 117.4, 116.5, 35.9, 23.8 ppm. HRMS: m/z calcd for
C20H21N4OS [M + H]+ 365.1431, found 365.1441; C20H20N4NaOS [M + Na]+
387.1250, found 387.1251.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-(phenylamino)phenyl)acetamide(14d)
Using the commercially available N1-phenylbenzene-1,4-diamine (12d), the title
compound 14d was obtained by general procedure 1 and general procedure 2 in turn,
1
48% for two steps, 96.8% HPLC purity. mp: > 300°C; H NMR (400 MHz, DMSO):
δ 10.08 (s, 1H),8.05 (s, 1H), 7.46 (d, J=8.8 Hz, 2H), 7.20 (t, J=8.0 Hz, 2H), 7.05-6.98
(m, 5H), 6.77 (t, J=7.2 Hz, 1H), 4.02 (s, 2H), 2.35 (s, 6H) ppm. 13C NMR (100 MHz,
DMSO): δ 169.8, 167.4, 166.3, 144.5, 139.4, 132.3, 129.6, 121.0, 119.5, 118.3, 116.5,
116.3, 35.9, 23.8 ppm. HRMS: m/z calcd for C20H21N4OS [M + H]+ 365.1431, found
365.1442; C20H20N4NaOS [M + Na]+ 387.1250, found 387.1251.
N-(4-benzylphenyl)-2-((4,6-dimethylpyrimidin-2-yl)thio)acetamide (14f) To a
mixture of LiAlH4 (10.0 equiv) in Et2O (5ml/mmol) was added the solution of AlCl3
(10.0 equiv) in Et2O (10ml/mmol) slowly at 0°C , and the reaction mixture was stirred
for 5 min. Then the solution of (4-aminophenyl)(phenyl)methanone (12h, 1.0 equiv )
in Et2O (15ml/mmol) was added, and the mixture was stirred at room temperature for
3 h. After completion (monitored by TLC), the mixture was diluted using 6M HCl and
neutralized by saturated sodium bicarbonate solution. Next, the solution was
partitioned between water and ethyl acetate (3×). The organic layer was dried over
magnesium sulfate anhydrous, filtered and concentrated in vacuo. The solid was
15

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purified by column chromatography to give the key intermediate 12f in 68% yield
(Scheme S1). Using 12f, the title compound 14f was obtained by general procedure 1
1
and general procedure 2 in turn, 53% for two steps, H NMR (400 MHz, DMSO):
δ10.17(s, 1H),7.49 (d, J=8.4 Hz, 2H), 7.28 (t, J=7.6 Hz, 2H), 7.22-7.15 (m, 5H), 6.97
(s, 1H), 4.02 (s, 2H), 3.89 (s, 2H), 2.34 (s, 6H) ppm. 13C NMR (100 MHz, DMSO): δ
169.8, 167.4, 166.8, 141.9, 137.5, 136.7, 129.4, 129.1, 128.8, 125.4, 119.8, 116.5,
40.0, 35.9, 23.8 ppm. HRMS: m/z calcd for C20H21N4OS [M + H]+ 364.1516, found
364.1513; C21H21N3NaOS [M + Na]+ 386.1298, found 386.1295.
N-(3-benzoylphenyl)-2-((4,6-dimethylpyrimidin-2-yl)thio)acetamide (14g) Using
the commercially available(3-aminophenyl)(phenyl)methanone (12g), the title
compound 14g was obtained by general procedure 1 and general procedure 2 in turn,
1
45% for two steps, 96.9% HPLC purity. H NMR (400 MHz, CDCl3): δ9.84(s, 1H),
8.02 (t, J=8.0 Hz, 1H), 7.80 (d, J=8.4 Hz, 2H), 7.66 (s, 1H), 7.62 (t, J=7.2 Hz, 1H),
7.52-7.43 (m, 4H), 6.85 (s, 1H), 3.91 (s, 2H), 2.50 (s, 6H) ppm. 13C NMR (100 MHz,
DMSO): δ 196.0, 169.7, 167.4, 139.6, 138.0, 137.5, 133.1, 130.0, 129.6, 129.0, 124.9,
123.5, 120.6, 116.5, 35.9, 23.8 ppm. LCMS m/z: 378.1 [M+H] +.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-(hydroxy(phenyl)methyl)phenyl)aceta
mide (14j) Firstly, according to the synthesis method for compound 12f, using 5.0
equiv LiAlH4 and AlCl3 the intermediate 12j was synthesized in 73% yield (Scheme
S1); Then, using 12j, the target compound 14j was got by general procedure 1 and
1
general procedure 2 in turn, 55% for two steps, 97.6% HPLC purity. H NMR (400
MHz, DMSO): δ 10.20(s, 1H),7.57 (s, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.36 (d, J=7.2 Hz,
16

ACCEPTED MANUSCRIPT
2H), 7.30 (t, J=8.0 Hz, 2H), 7.25-7.19 (m, 2H), 7.07(d, J=7.6 Hz, 1H), 6.97 (s, 1H),
13
5.89 (d, J=4.0 Hz, 1H), 5.65 (d, J=4.0 Hz, 1H), 4.01 (s, 2H), 2.32 (s, 6H) ppm. C
NMR (100 MHz, DMSO): δ 169.8, 167.4, 166.9, 146.9, 146.0, 140.2, 128.9, 128.3,
127.2, 125.3, 121.8, 119.9, 118.1, 117.6, 116.5, 74.8, 36.0, 23.8 ppm. HRMS: m/z
calcd for C21H22N3O2S [M + H]+ 380.1427, found 380.1435; C21H21N3NaO2S [M +
Na]+ 402.1247, found 402.1257.
General procedure 3: m-CPBA-involved oxidation reaction
To a solution of 2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-phenoxyphenyl)acetamide
(14b, 1.0 equiv)
in DCM
(20ml/1mmol) at
0°C
was
added
3-chlorobenzenecarboperoxoic acid (m-CPBA, 1.05 equiv), then the mixture was
allowed to warm to room temperature and stirred for 45 min. The resulting solution
was concentrated under reduced pressure to dryness and the residue was purified by
silica gel column chromatography to give the white finial compounds 15a in 62%
1
yield, 97.3% HPLC purity. mp: > 300°C; H NMR (400 MHz, CDCl3): δ 9.32(s,
1H),7.47 (d, J=8.8 Hz, 2H), 7.33 (t, J=7.6 Hz, 2H), 7.10 (t, J=8.8 Hz, 2H), 7.00-6.93
(m, 5H), 4.16 (d, J=14.4 Hz, 1H), 3.93 (d, J=14.4 Hz, 1H), 2.55 (s, 6H) ppm.13C
NMR (100 MHz, DMSO): δ 171.5, 168.9, 162.9, 157.7, 152.7, 134.9, 130.4, 123.5,
121.9, 121.4, 119.9, 118.4, 60.5, 23.9 ppm. HRMS: m/z calcd for C20H19N3NaO3S [M
+ Na]+ 404.1039, found 404.1030.
Similarly, to a solution of compounds 14b (1.0 equiv) in DCM (20ml/1mmol) at
0°C was added 3-chlorobenzenecarboperoxoic acid (m-CPBA, 3.0 equiv), then the
mixture was allowed to warm to room temperature and stirred for 3h. Upon
17

ACCEPTED MANUSCRIPT
completion of the reaction as determined by TLC, the resulting solution was
concentrated under reduced pressure to dryness and the residue was purified by silica
gel column chromatography to give the white finial compounds 15b in 71% yield,
97.6% HPLC purity.mp: > 300°C; 1H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H),7.48 (d,
J=8.8 Hz, 2H), 7.35 (t, J=8.0 Hz, 2H), 7.28 (s, 1H), 7.12 (t, J=8.8 Hz, 1H), 7.01-6.98
(m, 4H), 4.56 (s, 2H), 2.63 (s, 6H) ppm. 13C NMR (100 MHz, DMSO): δ 169.3, 164.5,
159.8, 157.6, 152.9, 134.6, 130.5, 123.7, 123.6, 121.4, 119.9, 118.5, 57.9, 23.8 ppm.
HRMS: m/z calcd for C20H20N3O4S [M + H]+ 398.1169, found 398.1159;
C20H19N3NaO4S [M + Na]+ 420.0988, found 420.0980.
General procedure 4: Hantzsch-involved reductive amination
Catalytic amount of molecular sieve and trifluoroacetic acid were added to the
solution of substituted anilins (1.0 equiv), different aromaticaldehydes (1.2 equiv),
and diethyl 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (Hantzsch, 1.2 equiv)
in CH2Cl2 at room temperature, and the reaction was warmed to 45°C and reacted for
12 h. After completion (monitored by TLC), the reaction was filtered, and the crude
residue was obtained by concentrating the filtrate in vacuo. Finally, the crude residue
was purified by column chromatography to give the intermediate or target compounds
in high yield.
General procedure 5: Fe-mediatedreduction reaction
The nitro compounds (1.0 equiv) were taken in EtOH (6 mL/mmol), and iron powder
(5.0 equiv) was added at 50°C-55 °C followed by NH4Cl solution (0.5 equiv in 3
mL/mmol water). The reaction mixture was refluxed for about 1 h[33,34]. When TLC
18

ACCEPTED MANUSCRIPT
indicated that the reaction was finished, most of the iron powder was filtered while
hot and the alcohol was removed under reduced pressure. The residue was basified
with NaHCO3 solution (pH 7-8) and extracted several times with ethyl acetate, the
combined organic extracts were dried (Na2SO4), and concentrated under reduced
pressure to yield the reduction product. These materials were taken up for the next
step without any purification.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-((thiophen-2-ylmethyl)amino)phenyl)a
cetamide (19b) The title compound was obtained by the general procedures as above,
1
19% for four steps, 96.7% HPLC purity. H NMR (400 MHz, DMSO): δ 9.80(s,
1H),7.32 (d, J=3.2 Hz, 1H), 7.24 (d, J=5.6 Hz, 2H), 7.01 (s, 1H), 6.93 (t, J=3.2 Hz,
2H), 6.57 (d, J=5.6 Hz, 2H), 6.07 (br s, 1H,), 4.39 (s, 2H ), 3.95 (s, 2H), 2.32 (s, 6H)
ppm. 13C NMR (100 MHz, DMSO): δ 170.8, 169.9, 167.4, 165.9, 145.1, 144.8, 129.2,
127.2, 125.2, 124.8, 121.3, 116.5, 113.0, 55.4, 35.8, 23.8 ppm. LCMS m/z: 385.1
[M+H] +.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-((pyridin-4-ylmethyl)amino)phenyl)ac
etamide (19f) The title compound was obtained by the general procedures as above,
21% for three steps, 96.6% HPLC purity. 1H NMR (400 MHz, DMSO): δ 9.83(s, 1H),
8.48 (d, J=5.6 Hz, 2H), 7.33 (d, J=6.0 Hz, 2H), 7.24 (d, J=8.8 Hz, 2H), 6.97 (s, 1H),
6.50 (d, J=8.8 Hz, 2H), 6.29 (t, J=6.0 Hz, 1H), 4.29 (d, J=6.4 Hz, 2H ), 3.96 (s, 2H),
13
2.32 (s, 6H) ppm. C NMR (100 MHz, DMSO): δ169.9, 167.4, 166.0, 150.2, 149.9,
145.1, 129.0, 122.8, 121.4, 116.5, 112.7, 46.1, 35.7, 23.8 ppm. LCMS m/z: 380.2
[M+H] +.
19

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2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(3-((phenylamino)methyl)phenyl)acetami
de (24a) The title compound was obtained by the general procedures as above, 30%
1
for four steps, 96.9% HPLC purity. mp: > 300°C; H NMR (400 MHz, DMSO): δ
10.21(s, 1H),7.55 (s, 1H), 7.48 (d, J=8.4 Hz, 1H), 7.25 (t, J=8.0 Hz, 1H), 7.06-7.01
(m, 3H), 6.96 (s, 1H), 6.55-6.49 (m, 3H), 6.24 (d, J=6.0 Hz, 1H), 4.22 (d, J=6.0 Hz,
13
2H), 4.02 (s, 2H), 2.32 (s, 6H) ppm. C NMR (100 MHz, DMSO): δ 169.8, 167.4,
166.9, 149.1, 141.6, 139.6, 129.3, 129.1, 122.6, 118.2, 118.0, 116.5, 116.2, 112.7, 47.0,
35.9, 23.8 ppm. LCMS m/z: 379.2 [M+H] +.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(3-(phenoxymethyl)phenyl)acetamide
(28a) 1-(Bromomethyl)-3-nitrobenzene (1.0 equiv) and phenol (1.05 equiv) were
dissolved in DMF (2ml/mmol) and reacted at room temperature for 12h, in the
presence of K2CO3 (3.0 equiv). After water (50ml/mmol) was added, the mixture was
partitioned between water and ethyl acetate (3×). The organic layer was dried over
magnesium sulfate anhydrous and concentrated in vacuo. The residue was purified by
column chromatography (eluent gradient PE/EA= 8/1) to give the intermediate 26a in
82% yield. Using 26a, the title compound was obtained by the general procedures as
1
above, 27% for four steps, 97.7% HPLC purity. mp: > 300°C; H NMR (400 MHz,
DMSO): δ 10.29(s, 1H), 7.69 (s, 1H), 7.54 (d, J=8.4 Hz, 1H), 7.35-7.28 (m, 3H), 7.14
(d, J=7.6 Hz, 1H), 7.01-6.93 (m, 4H), 5.08 (s, 2H), 4.04 (s, 2H), 2.33 (s, 6H)ppm. 13C
NMR (100 MHz, DMSO): δ 169.8, 167.4, 167.1, 158.7, 139.7, 138.3, 130.0, 129.3,
122.9, 121.2, 119.0, 118.5, 116.5, 115.2, 69.5, 35.9, 23.8 ppm. HRMS: m/z calcd for
C21H22N3O2S [M + H]+ 380.1427, found 380.1441; C21H21N3NaO2S [M + Na]+
20

ACCEPTED MANUSCRIPT
402.1274, found 402.1259.
2-((4,6-dimethylpyrimidin-2-yl)thio)-N-(4-(phenoxymethyl)phenyl)acetamide
(28b) The target compound was obtained using a method similar to that for 28a, 26%
for four steps, 96.3% HPLC purity. 1H NMR (400 MHz, DMSO): δ 10.30(s, 1H), 7.61
(d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.30 (t, J=7.2 Hz, 2H), 7.01-6.98 (m, 2H),
13
6.94 (t, J=7.2 Hz, 1H), 5.04 (s, 2H), 4.05 (s, 2H), 2.34 (s, 6H)ppm. C NMR (100
MHz, DMSO): δ 169.8, 167.4, 166.8, 149.2, 138.3, 135.5, 129.1, 128.3, 119.6, 116.5,
116.2, 112.7, 46.7, 35.9, 23.8 ppm. HRMS: m/z calcd for C21H22N3O2S [M + H]+
380.1428, found 380.1441; C21H21N3NaO2S [M + Na]+ 402.1274, found 402.1258.
N-(4-((3-(2-((4,6-dimethylpyrimidin-2-yl)thio)acetamido)benzyl)oxy)phenyl)thiop
hene-2-carboxamide (28e) First, N-(4-hydroxyphenyl)acetamide (1.05 equiv) react
with 1-(bromomethyl)-3-nitrobenzene (1.0 equiv) to give the intermediate 26c by the
methods describe as prepared for 26b; Second, to a solution of 26c (1.0 equiv) in
methanol (5ml/mmol) was added thionyl chloride (0.5ml/mmol) and the mixture was
reflux for 0.5h. Then the reaction was concentrated in vacuo to remove the organic
solvent and basified with NaHCO3solution (pH 8) and extracted three times with ethyl
acetate, the combined organic extracts were dried (Na2SO4), and concentrated under
reduced pressure to give 4-((4-nitrobenzyl)oxy)aniline, which was taken up for the
next step without any purification; Next, to a solution of 4-((4-nitrobenzyl)oxy)aniline
(1.0 equiv) and Et3N (3.0 equiv) in DCM (5ml/mmol) was added
thiophene-2-carbonyl chloride (1.2 equiv) at 0°C, and the reaction was stirred for 2h.
When TLC indicated that the reaction was finished, the reaction solution was
21

ACCEPTED MANUSCRIPT
concentrated under reduced pressure and the crude product was purified by column
chromatography (petroleum ether: EtOAc=4:1) to yield light yellow intermediate 26e.
Finally, using the key intermediate 26e, the title compound 28e was obtained by the
1
general procedures as above, 18% for six steps, 97.0% HPLC purity. H NMR (400
MHz, DMSO): δ 10.30(s, 1H), 10.13(s, 1H), 7.99 (d, J=3.2 Hz, 1H), 7.84 (dd, J=0.8
Hz, J=4.0 Hz, 1H), 7.71 (s, 1H), 7.63 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz, 1H), 7.34
(t, J=8.0 Hz, 1H), 7.22 (t, J=8.0 Hz, 1H), 7.14 (d, J=8.0 Hz, 1H), 7.02-6.97 (m, 3H),
13
5.08 (s, 2H), 4.06 (s, 2H), 2.36 (s, 6H)ppm. C NMR (100 MHz, DMSO): δ 169.8,
167.4, 167.1, 160.0, 155.1, 140.7, 139.7, 138.3, 132.4, 132.0, 129.2, 128.5, 122.9,
122.5, 119.0, 118.5, 116.5, 115.3, 69.8, 36.0, 23.8 ppm. HRMS: m/z calcd for
C26H25N4O3S2 [M + H]+ 505.1348, found 505.1369; C26H24N4NaO3S2 [M + Na]+
527.1179, found 527.1181.
N-(3-(((1H-indazol-5-yl)oxy)methyl)phenyl)-2-((4,6-dimethylpyrimidin-2-yl)thio)
acetamide (28k) Using the commercially available1H-indazol-5-ol, the title
compound 28k was obtained using a method similar to that for 28a, 17% for four
1
steps, 96.6% HPLC purity. H NMR (400 MHz, DMSO): δ 12.93 (s, 1H), 10.30 (s,
1H), 7.95 (s, 1H), 7.73 (s, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.34 (t,
J=8.0 Hz, 1H), 7.26 (s, 1H), 7.17 (d, J=8.0 Hz, 1H), 7.08 (dd, J=8.8 Hz, J=2.0 Hz,
13
1H), 6.96 (s, 1H), 5.11 (s, 2H), 4.05 (s, 2H), 2.32 (s, 6H)ppm. C NMR (100 MHz,
DMSO): δ 169.8, 167.4, 167.1, 153.2, 139.7, 138.5, 136.3, 133.3, 129.3, 123.5, 122.9,
118.9, 118.8, 118.5, 116.5, 111.6, 101.9, 70.1, 36.0, 23.8 ppm. HRMS: m/z calcd for
C22H22N5O2S [M + H]+ 420.1489, found 420.1490; C22H21N5NaO2S [M + Na]+
22

ACCEPTED MANUSCRIPT
442.1308, found 442.1313.
N-(3-(benzyloxy)phenyl)-2-((4,6-dimethylpyrimidin-2-yl)thio)acetamide (32) A
mixture of 3-aminophenol (29, 1.0 equiv), acetic anhydride (1.1 equiv), and
triethylamine (3.0 equiv) in DCM (10ml/mmol)was stirred at room temperature
overnight and the solvent was removed under reduced pressure. The solid residue was
partitioned between EtOAc and saturated aqueous NaHCO3, and the organic phase
was dried (Na2SO4). The solvent was removed in vacuo to afford
theN-(3-hydroxyphenyl)acetamide in 97% yield. Next, a reaction mixture of
N-(3-hydroxyphenyl)acetamide (1.0 equiv) and t-BuOK (1.5 equiv) in DMF
(5ml/mmol) was stirred for 0.5h at ambient temperature, and the resultant solution
was treated with benzylbromide (1.1 equivl) and the reaction mixture was stirred
overnight. After the reaction mixture was poured into a large amount of water
(20ml/1ml DMF), the crude mixture was extracted with ethyl acetate (3×). Then, the
combined organic layers were dried and concentrated. The residue was purified by
column chromatography to give intermediate 31, in 64% yield. Finally, using the key
intermediate 31, the title compound 32 was obtained by the general procedures as
1
above, 23% for five steps, 97.3% HPLC purity. H NMR (400 MHz, DMSO): δ
10.22(s, 1H),7.46-7.33 (m, 6H), 7.21 (d, J=8.4 Hz, 1H), 7.12 (d, J=8.4 Hz, 1H), 6.98
(s, 1H), 6.72 (dd, J=2.4 Hz, J=5.6 Hz, 1H), 5.07 (s, 2H), 4.04 (s, 2H), 2.34 (s, 6H)
ppm. HRMS: m/z calcd for C21H22N3O2S [M + H]+ 380.1427, found 380.1441;
C21H21N3NaO2S [M + Na]+ 402.1274, found 402.1259.
N-(4-(benzyloxy)phenyl)-2-((4,6-dimethylpyrimidin-2-yl)thio)acetamide (35) A
23

ACCEPTED MANUSCRIPT
reaction mixture of 1-fluoro-4-nitrobenzene (1.0 equiv), phenylmethanol (1.0 equiv)
and KOH(3.0 equiv) in dioxane (6ml/mmol)was stirred at room temperature over
night. Then the solvent was removed under reduced pressure and the solid residue was
partitioned between water and EtOAc, and the organic phase was dried (Na2SO4),
concentrated in vacuo. Next, the crude product was purified by column
chromatography
to
yield1-(benzyloxy)-4-nitrobenzene.
Finally,
using
1-(benzyloxy)-4-nitrobenzene, the title compound was obtained by the general
1
procedures described as above, 29% for four steps, 97.7% HPLC purity. H NMR
(400 MHz, DMSO): δ 10.10(s, 1H),7.49 (d, J=6.8 Hz, 2H), 7.46-7.44 (m, 2H),
7.41-7.38 (m, 2H), 7.35-7.31(m, 1H), 7.00-6.95 (m, 3H), 5.07 (s, 2H ), 4.01 (s, 2H),
2.34 (s, 6H) ppm. 13C NMR (100 MHz, DMSO): δ 169.8, 167.4, 166.5, 154.8, 137.6,
132.9, 128.9, 128.2, 128.1, 121.1, 116.5, 115.3, 69.8, 35.8, 23.8 ppm. HRMS: m/z
calcd for C21H22N3O2S [M + H]+ 380.1427, found 380.1442;C21H21N3NaO2S [M +
Na]+ 402.1274, found 402.1260.
Molecular Docking Simulations
In this study, all the docking simulations were carried out using AutoDock
Vina[35]. The crystal structure of SIRT2 complexed with SirReal2 (PDB ID:
4RMG)[29] was used as the docking template. All the water and solvant molecules as
well as SirReal2 were removed, and NAD+ was kept. Gasteiger-Marsili charges were
added to protein model and non-polar hydrogens were merged onto their respective
heavy atoms using AutoDockTools. The binding site is defined as a rectangular grid.
The grid center was set as coordinates of [x, y, z=-16.0, -26.1, 11.8], and the grid size
24

ACCEPTED MANUSCRIPT
was 22×25×23, which encompassed the entire SirReal2 binding site. The number of
docking poses was set as 20, and other parameters were set as default. Docking results
were viewed using PyMOL.
Cell Line and Cell Culture
Human MCF-7 breast carcinoma cells were purchased from American
TypeCulture Collection (ATCC, Rockville, MD) and maintained in DMEM (corning,
High Glucose) medium supplement with 10% FBS (cell box), 100
U/mlpenicillin(Sigma–Aldrich, USA)and 100 U/ml streptomycin(Sigma–Aldrich,
USA).
Cell Growth Inhibition Assays
The MTT assay was used to detect the viability of cells. Human MCF-7 breast
carcinoma cells were subcultured at 80~90% confluency and used within 10 passages.
First, cells were collected and seeded in a 96-well plate, and the seeding densities
were 3000 cells/well. Then, cells were grown in media (10% FBS) in 96-well plates
for 24 h, at 37℃, 5% CO2. Next, different concentrations of test compounds (stock
solution in DMSO, diluted with media containing 10% FBS) were added to each well
and were incubated for 120 h, at 37℃, 5% CO2. Finally, concentration of DMSO per
well was 1% v/v. At the end of the incubation period, 20µL of the MTT (5mg/ml,
Sigma, USA) solution was added to each well. And then, the formazan crystals were
dissolved with 50µL of acidified SDS (20%, w/v). Absorbance was determined at
570nm on Multiskan MK3 (Thermo Scientific, USA) the next day. Each assay was
performed in three replicates and all experiments were repeated at least twice.
Western Blotting Analyses
25

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The MCF-7 cells were plated to 6-well plates (Corning) at a density of 1*105
cells/well, and the experiments were initiated after 24 h. The cells were treated with
vehicle (0.5% DMSO) or compound 28e for 6 h. MCF-7 cells were then harvested,
washed with ice-cold physiological saline, and lysed with RIPAlysis buffer (Beyotime,
China) containing 1% cocktail (Sigma-Aldrich)[19,36]. Proteins were separated by
gel electrophoresis on 10% SDS-PAGE gels and probed with specific antibodies used
at a 1:1,000 dilution. Then anti-α-tubulin (T6074) and anti-α-tubulin acetylated
(T6793) of mouse monoclonal antibodies were both purchased from Sigma Aldrich.
The horse radish peroxidase-coupled secondary antibodies (Zhong Shan Golden
Bridge Bio-technology, China) were used at 1:5,000.
Acknowledgements
This work was supported by Talent Introduction Key Project of Xihua University
(No. z1410526),
National Natural Science Foundation of China
Undergraduate Innovation and entrepreneurship
(No.201510623048).
Appendix. Supplementary material
Sichuan Education Department Key Projects
(No. 81502989)
training
(No.16205459),
and
National
program
Supplementary data related to this article can be found online at doi:10.1000/j.ejmech.
0000.00.000.
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