A new class of 1,3,5-triazine-based selective estrogen receptor degraders (SERDs): Lead optimization, molecular docking and dynamic simulation

Article abstract of DOI:10.1016/j.bioorg.2020.103666

Selective estrogen receptor degrader (SERD) that acts as not only ER antagonist, but also ER degrader, would be useful for the treatment for drug-resistance ER+ breast cancer. However, most of currently available SERD candidates involve very limited molecular scaffolds and are still in clinical trials. In this study, we introduced a 1,3,5-triazine ring into a homobibenzyl motif extracted from amounts of ER ligands and synthesized sixteen SERDs bearing acrylic acid or acrylic amide side chains that possess both ERα antagonism and degradation properties. And all compounds were screened for their anti-proliferative activity against ER+ MCF-7 and Ishikawa cell lines. Among them, compound XHA1614 displayed potent growth inhibition activity against MCF-7 and Ishikawa cells with IC₅₀ values of 3.15 μM and 3.11 μM, respectively. Moreover, XHA1614 could dramatically degrade ER level at 1 nM in a Western blotting assay and afforded an outstanding antagonistic activity via suppressing the expression of progesterone receptor messenger RNA in MCF-7 cells in a RT-PCR assay. Further molecular docking and dynamic simulation on properly selected derivative furnished insights into its binding profile within ERα. Our findings suggest that the 1,3,5-triazine core was a feasible alternative to currently reported SERD scaffold, and provide information that will be useful for further development of promising SERDs candidates for breast cancer therapies.

Full text of DOI:10.1016/j.bioorg.2020.103666

BioorganicChemistry97(2020)103666
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A new class of 1,3,5-triazine-based selective estrogen receptor degraders
(SERDs): Lead optimization, molecular docking and dynamic simulation
⁎
Xiang Lua,b, Ali Huanga,b, Maoxu Xiaoa,b, Liang Sunc, Jiashun Maoc, Guoshun Luoa,b,
,
⁎
Hua Xianga,b,
a State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c Shenzhen Shuli Tech Co., Ltd, Shenzhen 518126, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
1,3,5-triazine
ERα
Selective estrogen receptor degrader (SERD) that acts as not only ER antagonist, but also ER degrader, would be
useful for the treatment for drug-resistance ER+ breast cancer. However, most of currently available SERD
candidates involve very limited molecular scaffolds and are still in clinical trials. In this study, we introduced a
1,3,5-triazine ring into a homobibenzyl motif extracted from amounts of ER ligands and synthesized sixteen
SERDs bearing acrylic acid or acrylic amide side chains that possess both ERα antagonism and degradation
properties. And all compounds were screened for their anti-proliferative activity against ER+ MCF-7 and
Ishikawa cell lines. Among them, compound XHA1614 displayed potent growth inhibition activity against MCF-
7 and Ishikawa cells with IC50 values of 3.15 μM and 3.11 μM, respectively. Moreover, XHA1614 could dra-
matically degrade ER level at 1 nM in a Western blotting assay and afforded an outstanding antagonistic activity
via suppressing the expression of progesterone receptor messenger RNA in MCF-7 cells in a RT-PCR assay.
Further molecular docking and dynamic simulation on properly selected derivative furnished insights into its
binding profile within ERα. Our findings suggest that the 1,3,5-triazine core was a feasible alternative to cur-
rently reported SERD scaffold, and provide information that will be useful for further development of promising
SERDs candidates for breast cancer therapies.
SERDs
Breast cancer
1. Introduction
therapy before [7]. Based on this continued dependence on ER, there is
a need for novel endocrine treatments with improved efficacy that
Breast cancer is the most frequently occurring cancer and the second
leading cause of mortality in women [1]. Approximately 75% of all
breast cancers diagnosed are estrogen receptor positive (ER+) breast
cancer that can be effectively treated with antihormone therapy [2].
Tamoxifen (Fig. 1, 1) and its active metabolite 4-hydroxyTamoxifen
(Fig. 1, 2), two typical selective estrogen receptor modulators (SERMs),
act as antagonists of ER in breast tissue and induce tumor regression by
a blockade of estrogen signal [3]. Although patients with ER+ breast
cancer initially respond positively to this therapeutic drug, de novo and
acquired resistance often occur after long-term treatment [3–5]. There
are a variety of mechanisms that contribute to this phenomenon, of
note is that the estrogen receptor still plays a critical role in driving
tumor cells to grow in this state [6]. Moreover, the mutations of ESR1
that constitutively activate ERα without the need for hormone binding
are frequently found in refractory patients who received endocrine
could potentially treat patients with relapsed breast cancers.
Currently, Fulvestrant (Fig. 1, 3), a steroidal antiestrogen, is the
only approved selective estrogen receptor degrader (SERD) [8], being
able to not only directly antagonize ER function but also induce ER
degradation. This distinguished mechanism of receptor degradation has
been found to be clinically effective in breast cancer patients who have
progressed on prior endocrine agents [9,10]. Unfortunately, Fulvestrant
has poor oral bioavailability [11] and is administered only by in-
tramuscular injection [12]. And incomplete removal of ER in vivo
limited its clinical efficacy [13]. Recently, several orally bioavailable
SERDs possessing nonsteroidal scaffolds were reported in clinical de-
velopment, including LSZ-102 (4) [14], AZD9496 (5) [15], and SS5020
(6a) and its derivative (6b) [16,17]. Unlike SERMs bearing basic side
chains, however, the acrylic acid moieties of these SERDs repositioned
Helix-12 in an unusual acid-acid interaction, increasing the exposed
⁎ Corresponding authors at: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China.
E-mail addresses: gsluo@cpu.edu.cn (G. Luo), xianghua@cpu.edu.cn (H. Xiang).
https://doi.org/10.1016/j.bioorg.2020.103666
Received 14 August 2019; Received in revised form 1 February 2020; Accepted 12 February 2020
Availableonline13February2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

X. Lu, et al.
BioorganicChemistry97(2020)103666
Fig. 1. Chemical structures of certain estrogen receptor ligands.
hydrophobic surface of ERα resulting in destabilization and ultimately
proteasomal degradation of the receptor [18–20]. Despite these ad-
vancements, no oral SERD has yet been approved for marketing, thus
the therapeutic value of SERDs containing novel chemical scaffolds
with better drug-like properties remains to be further explored.
Rather than seeking to optimize known ER motifs, we chose to in-
vestigate a less explored heterocyclic core, such as 1,3,5-triazine. The
rational for this choice was also based on a “generic-core approach”
that the core element homobibenzyl motif found in numerous high af-
finity ER ligands can be employed to construct ER ligands by introdu-
cing heterocycles, such as previously reported pyrazole- and pyr-
imidine-based SERMs [21–23]. Herein, we reported the combination of
the 1,3,5-triazine core with phenyl acrylic acid moiety found in above-
mentioned SERDs (4–6) (Fig. 2) with the goal of identifying a new
chemo-type of SERDs that could then be optimized for cellular potency.
All compounds were tested by antiproliferative assay in MCF-7. More-
over, molecular dynamics simulation revealed that compound
XHA1613 demonstrated a similar binding model in ERα with original
ligand (6b). And in the further experiments, Western blotting and RT-
PCR, compound XHA1614 showed an outstanding potency compared
to Fulvestrant and Tamoxifen.
(XHA1601-XHA1605) were obtained from corresponding compounds
15–19 by hydrolyzation under base condition. Compounds XHA1601
and XHA1602 were demethylated with BBr3 to obtain compounds
XHA1607 and XHA1608 in high yields. Besides, acylation of com-
pounds XHA1601, XHA1607 and XHA1608 with 3-amino-1-propanol
using HATU as catalyst prepared compounds XHA1606, XHA1609 and
XHA1610 respectively [24].
For the synthesis of 2,4,6-triaryl-1,3,5-triazine derivatives
(XHA1611–XHA1616), it also started from commercially available
1,3,5-triazine (Scheme 3). This triazine ring reacted with 4-methox-
yphenylboronic acid via Suzuki coupling reaction for twice orderly,
giving the intermediate 26 in good yields [25]. Next, by using Suzuki
coupling reaction once more, 4-formylphenyl was introduced at 2-po-
sition of the core element to prepare compound 27. Then, compound 27
reacted with ethyl(triphenylphosphoranylidene)acetate in dried di-
chloromethane to yield compound 28. Next, compound 28 was trans-
ferred to XHA1611 by hydrolyzation which was converted to XHA1612
in similar synthetic way as the synthesis of XHA1609. Besides, com-
pound XHA1613 was prepared from XHA1611 by demethylation with
BBr3. Moreover, compound XHA1613 was acylated with three kinds of
aliphatic chain amine with terminal polar groups (eOH or eCF3) to get
XHA1614-XHA1616, which was catalyzed by HATU.
2. Results and discussions
3. Biological evaluation
2.1. Chemistry
3.1. Cell proliferation assay.
The synthesis of 4-aminobenzeneacrylic acid ethyl ester (11) is
described in scheme 1. p-Nitrobenzaldehyde (8) reacted with propa-
nedioic acid in pyridine at 100 °C to obtain 4-nitrocinnamic acid (9) in a
good yield. Then, the acid was converted to ethyl 4-nitrocinnamate (10)
readily with ethanol under catalysis of sulfuric acid. Finally, 4-amino-
benzeneacrylic acid ethyl ester (11) was obtained from ethyl 4-ni-
trocinnamate (10) by reduction with Fe/NH4Cl quantificationally.
The synthetic compounds were tested for potential cytotoxic effects
in MCF-7 (ER+) using MTT assay. Notably, it is well-known that a long-
term use of Tamoxifen will increase the risk of endometrial cancer in
patients [26,27], thus, it is necessary to evaluate antiproliferation ac-
tivity on human endometrial cancer cell line Ishikakwa. Besides, clin-
ical approval SERM Tamoxifen and SERD Fulvestrant were selected as
positive controls for comparison in this assay.
As
for
the
2-anilino-4,6-biaryl-1,3,5-triazine
analogs
XHA1601–XHA1610, the synthetic routes were showed in Scheme 2.
Nucleophilic substitution of commercially available 1,3,5-triazine (12)
with pre-synthesized side chain (11) yielded compound 13 to which
was introduced the first 4′- methoxyaryl via Suzuki coupling reaction,
giving the intermediate (14). Then, this intermediate reacted with
various substituted phenylboronic acid, resulting in reintroducing an-
other aryl group to afford compounds 15–19. Finally, the compounds
As shown in Table 1, the cell antiproliferation data can be analyzed
in three factors: the substituents on two phenyl which mimic the A/D
ring of estradiol; the side chains and two different linkers. Firstly, when
the para-position of 2-phenyl was methoxy group, the compound
XHA1601 with an electron-donating group (eOCH3) introduced at
para-position of 6-phenyl showed a better cell antiproliferation activity
than the other compounds which possessed electron withdrawing
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X. Lu, et al.
BioorganicChemistry97(2020)103666
Fig. 2. Reported ER ligands containing N-heterocycle scaffold and our designed 1,3,5-triazine-based SERDs.
there was a moderately rise in activity of Ishikawa cells illustrating that
all synthetic compounds did not act as agonists in endometrial cells,
while the cell antiproliferation activity of Tamoxifen in Ishikawa cells
was weaker than the activity in MCF-7 cells. Among these compounds,
XHA1614 had an outstanding cell antiproliferation activities in MCF-7
and Ishikawa (IC50 = 3.15 μM, 3.11 μM, respectively) which were
better than Tamoxifen. Thus, this compound was selected for further
bioactivity experiments.
Scheme 1. Synthetic routes of 4-aminobenzeneacrylic acid ethyl ester.
Reaction conditions and reagents: (a) malonic acid, pyridine, piperidine, reflux,
100 °C, 2 h, 82%; (b) ethanol, sulfuric acid, 70 °C, 3 h, 97%; (c) Fe, NH4Cl,
ethanol, H2O, 85 °C, 1 h, 100%.
3.2. Molecular docking studies and molecular dynamics simulation of
interaction between XHA1613 and ER
In order to investigate the feasible binding model and details of
interaction mechanism between synthetic compounds and ER, mole-
cular docking studies and molecular dynamics simulation were utilized
as useful tools to visually display the change of interaction between
receptor and ligand. We speculated that two hydroxy groups at the 2/6-
phenyl of those synthetic compounds might mimic those groups of es-
tradiol which were benefit for binding affinity. Thus, XHA1613 which
possessed the reported acrylic acid side chain was selected as the
sample.
groups (eCl, eF, eOCF3) at the same position (XHA1601 vs
XHA1603–XHA1605). Besides, compared to compound XHA1601,
there was a sharply decrease in activity of the corresponding hydroxy
compound XHA1607. Secondly, the amide side chain analogues had a
dramatically increase in activity compared to the corresponding acrylic
acid compounds (XHA1606 vs XHA1601, XHA1609 vs XHA1607,
XHA1612 vs XHA1611, XHA1614 vs XHA1613). And the amounts of
atoms of the amines line or what the polar groups were did not affect
the activity. Finally, the NH-linkers derivatives held a little weaker cell
antiproliferations activities than another series. And as for anti-
proliferation activities in Ishikawa, similar trends were also seen in this
cell line. What’s more, what difference of these two cell lines is that
Molecular docking studies was performed using Schrodinger 10.2/
Glide docking protocol. As shown in Fig. 3C, at a global view, there was
a remarkable overlap of compound XHA1613 and 6b (co-crystallized
ligand), which figured that these synthetic compounds showed the
same binding mode like that of 6b. Furthermore, in Fig. 3A and 3B, as
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X. Lu, et al.
BioorganicChemistry97(2020)103666
Scheme 2. Synthetic routes of compounds XHA1601–XHA1610. Reaction conditions and reagents: (a) Na2CO3, Acetone, H2O, 0–5 °C, 2 h, 76%; (b) Pd(PPh3)2Cl2,
K2CO3, Toluene, H2O, 60 °C, 6 h, 75%; (c) Pd(PPh3)2Cl2, K2CO3, Toluene, H2O, 100 °C, 6 h, 68–99%; (d) KOH, THF, Methanol, 60 °C, 3.5 h, 59–97%; (e) 3-
Aminopropanol, HATU, DIPEA, anhydrous DMF, rt, 5 h, 57–75%; (f) BBr3, anhydrous CH2Cl2, 0 °C, 6 h, 70–80%.
our speculation, the detail docking analysis illustrated that the hydroxyl
group at para-position of 2-phenyl of compound XHA1613 formed
hydrogen bonds with Glu353 and Arg394 like 6b which were the key
binding factor to ERα. Moreover, the terminal carboxylic acid group of
XHA1613 formed another hydrogen bond interaction with Val534
which was not exist in the binding model of 6b.
parameters, including binding free energy, root mean square deviation
(RMSD) and root mean square fluctuation (RMSF) were calculated to
reflect similarities and differences of docking model between XHA1613
and 6b. RMSD was utilized to evaluate stability of entire conformation
of ligand simulated by molecular dynamics. As shown in Fig. 4, from 0
to 10 ns, there was a slightly increase of RMSD value in XHA1613-ER
complexity system, while a sharply increase to about 3.5 Å was wit-
nessed in another system. Unlike the value fluctuated at 2.5 Å in
XHA1613-ER complexity system during 10–50 ns, the RMSD value of
For the further investigation of interaction mechanism of XHA1613
within ERα, two ligand-receptor complex systems (XHA1613-ER and
6b-ER) were constructed using molecular dynamics simulation. Three
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X. Lu, et al.
BioorganicChemistry97(2020)103666
Scheme 3. Synthetic routes of compounds XHA1611–XHA1616. Reaction conditions and reagents: (a) 4-Methoxyphenylboronic acid, Pd(PPh3)2Cl2, K2CO3,
Toluene, 60 °C, 12 h, 44%; (b) 4-Methoxyphenylboronic acid, Pd(PPh3)2Cl2, K2CO3, Toluene, H2O, 60 °C, 12 h, 67%; (c) 4-Formylphenylboronic acid, Pd(PPh3)2Cl2,
K2CO3, DMF, H2O, 100 °C, 6 h, 74%; (d) Ethyl(triphenylphosphoranylidene)acetate, anhydrous CH2Cl2; (e) KOH, THF, Methanol, 60 °C, 3.5 h, 84%; (f) RNH2, HATU,
DIPEA, anhydrous DMF, rt, 5 h, 67%.
6b-ER complexity system shook at 3.5 Å during this period. Thus, the
values of these two systems indicated that compound XHA1613 showed
weaker influence on receptor conformation than another system.
RMSF was used to evaluate the volatility of every amino acid re-
sidue during the period of stimulation. Obviously, there were several
fluctuations around amino acid residues 325–350, 450–475 and
525–530 in these two systems according to the values of RMSF (Fig. 5),
which indicated that these two systems held similar binding model.
Moreover, binding free energies illustrated that around amino acid
340–360 and 480–495 contributed to the binding of the ligands and
receptor which were consistence with the molecular binding studies
(Fig. 6). Thus, the binding free energies results further confirmed that
these two complexity systems possessed similar binding models for
same change trend of similar amino acid residues.
3.3. The inhibition activity of compound XHA1614 against the expression
of progesterone receptor mRNA in MCF-7 cells
Furtherly, quantitative real-time polymerase chain reaction (RT-
PCR) in MCF-7 (ER+) was adopted to investigate expression of pro-
gesterone receptor (PgR) of which the expression is normally associated
with estrogenic or antiestrogenic activity. It was illustrated in Fig. 7A
that at the presence of 10 nM estradiol (E2) the mRNA expression of
PgR gene was raised remarkably compared to vehicle. By contrast, as-
sociating 10 nM estradiol with compound XHA1614 at 1 μM con-
centration would dramatically decrease the expression of PgR gene,
which demonstrated that this compound possessed an attractive anti-
estrogenic property. Moreover, the tested compound presented equal
potency as two positive controls.
Moreover, compound XHA1614 was tested for the mRNA expres-
sion of progesterone receptor gene at five different does ranging from
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X. Lu, et al.
BioorganicChemistry97(2020)103666
Table 1
Antiproliferation activity of XHA1601–XHA1616.
Comp.
R1
R2
R3
Antiproliferation (IC50, μM)a
MCF-7
Ishikawa
XHA1601
XHA1602
XHA1603
XHA1604
XHA1605
XHA1606
XHA1607
XHA1608
XHA1609
XHA1610
XHA1611
XHA1612
XHA1613
XHA1614
XHA1615
XHA1616
Tamoxifen
Fulvestrant
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOCH3
eOH
eOCH3
eH
eOH
23.71
35.23
53.16
49.67
37.08
5.97
16.95
17.33
19.64
29.84
18.79
2.98
eOH
eCl
eOH
eF
eOH
eOCF3
eOCH3
eOH
eH
eOH
eNH(CH2)3OH
eOH
78.95
39.51
5.49
42.38
29.21
1.17
eOH
eOH
eOH
eOH
eH
eNH(CH2)3OH
eNH(CH2)3OH
eOH
eOH
10.11
60.37
16.80
60.37
3.15
1.68
eOCH3
eOCH3
eOH
eOCH3
eOCH3
eOH
eOH
eOH
eOH
–
26.11
4.06
eNH(CH2)3OH
eOH
22.95
3.11
eOH
eNH(CH2)3OH
eNH(CH2)2OH
eNHCH2CF3
–
eOH
4.00
——
eOH
3.87
——c
16.63
ND
–
10.69
NDb
–
–
–
a
b
c
IC50: concentration that inhibits 50% of cell growth.
ND: stands for not determined.
——: stands for not detected.
Fig. 3. (A) Docking interactions of XHA1613 within ERα binding pocket. (B) Docking interactions of 6b within ERα binding pocket. Red dashed line stands for
hydrogen bonds. (C) Covering of XHA1613 (light blue) and 6b (Yellow, PDB ID:5AK2).
6

X. Lu, et al.
BioorganicChemistry97(2020)103666
Fig. 6. Binding Free Energies of XHA1613-ER complexity system and 6b-ER
complexity system.
10-9 to 10-5 M. As shown in Fig. 7B, the compound XHA1614 inhibited
the expression of PgR gene in a dose-dependent manner.
3.4. The degrading activity of compound XHA1614 against ERα and Ki67
in MCF-7
Fig. 4. RMSD Values of XHA1613-ER complexity system and 6b-ER complexity
system.
A big difference between SERMs and SERDs is that the latter also
can degrade ERα. Additionally, Ki67 is a crucial tumor marker of var-
ious kinds of tumor cells. And it is believed that antagonism of ERα has
a relevance with a decrease of Ki67 [28]. Thus, Western blotting assay
was utilized to evaluate the expression of these two proteins. As shown
in Fig. 8, the compound XHA1614 dramatically reduced the expression
of ERα in MCF-7 at 10-9 M compared to vehicle, while Fulvestrant-the
positive control-could only decrease the expression of ERα at 10-6 M.
Obviously, the compound XHA1614 presented a strong degrading ac-
tivity in ERα than Fulvestrant. Furthermore, this compound XHA1614
has an ability to block the expression of Ki67 in a dose-dependent
manner, while Fulvestrant only depressed the expression of Ki67 at 10-6
M. According to the result of Ki67, the compound XHA1614 showed a
slightly better potency than the positive control.
Fig. 5. RMSF Values of XHA1613-ER complexity system and 6b-ER complexity
4. Conclusion
system.
In summary, we designed and synthesized a series of 2-cinnamic
acid-4,6-diaryl substituted 1,3,5-triazine derivatives to explore dual
activity of being transcriptional antagonists and degraders of the es-
trogen receptors (SERDs). Most of these compounds showed from
mildly to potent activities in cell antiproliferation in MCF-7 cells and
Ishikawa cells. And similar binding model as 6b was confirmed by
molecular docking studies and molecular dynamics simulation with
dihydroxy compound XHA1613. Moreover, among these synthetic
Fig. 7. (A) The increased mRNA expression of PR induced by E2 was reversed by XHA1614 in MCF-7 cells. Fulvestrant and Tamoxifen were positive controls. (B)
Compound XHA1614 was investigated at five different does ranging for 10-9–10-5 M. The mRNA expression of PR was examined by Real-time PCR. The mRNA
expression of PR was examined by Real-time PCR. Values are mean
SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.
7

X. Lu, et al.
BioorganicChemistry97(2020)103666
Fig. 8. (A) Western blotting analysis of ERα and Ki67 protein levels in MCF-7 cells treated with XHA1614. (B) Western blotting analysis of ERα and Ki67 protein
levels in MCF-7 cells treated with Fulvestrant. XHA1614 and Fulvestrant were tested at the concentrations of 10-9–10-6 M.
compounds, XHA1614, holding a remarkable cell antiproliferation ac-
tivity in MCF-7 and Ishikawa cells (IC50 = 3.15 μM, 3.11 μM, respec-
tively), was selected for the further evalution. In quantitative real-time
polymerase chain reaction (RT-PCR) assay, the compound XHA1614
and positive controls, Tamoxifen and Fulvestrant, presented attractive
inhibition potency of expression of PgR gene equally and compound
XHA1614 inhibited the expression of PgR gene at a dose-dependent
manner, indicating XHA1614 could be regarded as an antagonist of
ERα like Tamoxifen. Moreover, analyzing the effect of XHA1614 and
Fulvestrant on ERα and Ki67 levels using Western blotting, this com-
pound reduced levels of ERα to a higher extent than Fulvestrant, con-
forming XHA1614 was a strong degrader of ERα. Besides, the potency
of decreasing levels of Ki67 protein of XHA1614 was also slightly
higher than Fulvestrant. All in all, our results documented that the
newly developed 1,3,5-triazine-core derivatives not only showed si-
milar binding model like 6b and crucial anti-cancer activity, but pos-
sessed attractive antagonism effect of ERα and remarkable potency of
degrading of ERα, which was regarded as a lead compound and worth
to be modified in the next further step.
was stirred at room temperature for 10 min. Then, the corresponding
amine was added dropwise into the solution. And the resulting yellow
solution was stirred at room temperature for another 20 min. After
then, N,N-diisopropylethylamine (0.6 mmol) was added by syringe and
the final mixture was stirred at room temperature for additional 5 h.
After completion of the reaction, the reaction mixture was poured into
ice water and filtered. And the residue was recrystallized by tetra-
hydrofuran/petrol ether to obtain product.
5.1.4. General procedure C: Demethylation
A solution of boron tribromide (4.4 mmol) in dried dichloromethane
(1.45 ml) was added to a solution of compound (0.44 mmol) in dried
dichloromethane (9 ml) at 0 °C under nitrogen condition. The mixture
was stirred at room temperature for 6 h. After completed, the mixture
was poured into ice water carefully, adjusted to pH 5–6 by 1 M NaOH
and concentrated in vacuo. And the residue was redissolved in ethyl
acetate and washed with brine. The organic layer was combined, dried
over Na2SO4 and concentrated in vacuum. The crude product was re-
crystallized by petrol ether/ ethyl acetate.
5. Experimental section
5.1.5. (E)-3-(4-nitrophenyl) acrylic acid (9)
4-nitrobenzaldehyde (5 g, 33 mmol), carboxyacetic acid (5.15 g,
49.50 mmol) and piperidine (3 ml, 33 mmol) were dissolved in pyridine
(30 ml). And the mixture was stirred at 100 °C for 2 h. After completion
of the reaction, the mixture cooled to room temperature and was
poured into ice water, acidized by 6 N HCl and filtered, giving pale
yellow solid (5.26 g, 82%). 1H NMR (300 MHz, DMSO‑d6) δ: 12.69 (s,
1H), 8.22 (d, J = 8.7 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.68 (d,
J = 15.9 Hz, 1H), 6.73 (d, J = 16.0 Hz, 1H).
5.1. Chemistry
5.1.1. General
Most chemicals and solvents were of analytical grade and, when
necessary, were purified and dried by standard methods. Reactions
were monitored by thin-layer chromatography (TLC) using precoated
silica gel plates (silica gel GF/UV 254), and spots were visualized under
UV light (254 nm). Melting points (uncorrected) were determined on a
Mel-TEMP II melting point apparatus and are uncorrected. 1H NMR and
13C NMR spectra were recorded with a Bruker Avance 300 MHz/
400 MHz spectrometer at 300 K, using TMS as an internal standard. MS
spectra were recorded on a Shimadzu GC–MS 2050 (ESI) or an Agilent
1946A-MSD (ESI) Mass Spectrum. Column chromatography was per-
formed with silica gel (200–300 mesh). Chemical shifts (d) are ex-
pressed in parts per million relative to tetramethylsilane, which was
used as an internal standard, coupling constants (J) are in hertz (Hz),
and the signals are designated as follows: s, singlet; d, doublet; t, triplet;
m, multiplet.
5.1.6. (E)-3-(4-nitrophenyl) acrylate ethyl (10)
Concentrated sulfuric acid (5 ml) was added to a solution of 9 (5 g,
25.89 mmol) in 100 ml ethanol. The mixture was stirred at 70 °C for
3 h. After consumption of start material, the reaction mixture cooled to
room temperature and was poured into ice water and filtered, obtaining
yellow solid (5.60 g, 97%). 1H NMR (300 MHz, CDCl3) δ: 8.20 (d,
J = 8.7 Hz, 2H), 7.66 (dd, J = 16.1, 8.7 Hz, 3H), 6.53 (d, J = 16.1 Hz,
1H), 4.25 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H).
5.1.7. (E)-ethyl 3-(4-aminophenyl) acrylate (11)
5.1.2. General procedure A: Hydrolysis of ethyl ester
A suspended of 10 (2.31 g, 10.44 mmol) in a mixture of EtOH
(35 ml) and H2O (9 ml) was added Iron powder (1.75 g, 31.32 mmol)
and ammonium chloride (0.29 g, 5.43 mmol). The suspension was
stirred at 85 °C for 1 h. After completed of the reaction, the mixture was
filtered through a pad of Celite and the filter cake was washed by 1:1
EtOH/DCM (250 ml). Then the filtrate was concentrated in vacuo to
remove organic layers, redissolved in ethyl acetate and washed with
water, brine. And the organic layers were dried with Na2SO4 and
concentrated in vacuum to yield yellow solid (2.10 g, 100%). 1H NMR
(300 MHz, CDCl3) δ: 7.61 (d, J = 15.9 Hz, 1H), 7.35 (d, J = 8.4 Hz,
2H), 6.65 (d, J = 8.4 Hz, 2H), 6.24 (d, J = 15.9 Hz, 1H), 4.25 (q,
J = 7.1 Hz, 2H), 3.98 (s, 2H), 1.33 (t, J = 7.1 Hz, 3H).
A solution of potassium hydroxide (2 mmol) in methanol (2 ml) was
added to a solution of the ester (0.4 mmol) in tetrahydrofuran (6 ml).
The resulting mixture was stirred at 60 °C for 3.5 h. After completed,
the mixture was poured into ice water, acidized to pH 4–5 by 1 N HCl
and filtered. And the residue was recrystallized by dichloromethane to
obtain product.
5.1.3. General procedure B: Amide synthesis using HATU
The mixture of compound (0.2 mmol) and O-(7-;2-(7-
Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoropho-
sphate (HATU, 0.3 mmol) in dried N,N-dimethylformamide (1.5 ml)
8

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BioorganicChemistry97(2020)103666
5.1.8. (E)-ethyl
3-(4-((4,6-dichloro-1,3,5-triazine-2-yl)amino)phenyl)
J = 9.1 Hz, 2H), 6.41 (d, J = 16.1 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H),
acrylate (13)
3.91 (s, 3H), 1.35 (t, J = 7.0 Hz, 3H).
A solution of 11 (0.52 g, 0.71 mmol) in acetone (5.2 ml) and 20%
sodium carbonate aqueous solution (0.29 g, 2.71 mmol) were added to
a solution of 1,3,5-triazine (12, 0.50 g, 2.71 mmol) in acetone (5 ml)
slowly at 0–5 °C over 2 h. After that, the mixture was poured in water
and filtered, giving crude product. And the crude product was re-
crystallized with acetone to obtain compound 13 (0.76 g, 76%). 1H
NMR (300 MHz, CDCl3) δ: 7.61 (dt, J = 14.9, 8.7 Hz, 6H), 6.41 (d,
J = 16.0 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).
5.1.13. (E)-ethyl 3-(4-((4-(4-fluorophenyl)-6-(4-methoxyphenyl)-1,3,5-
triazin-2-yl)amino)phenyl)acrylate (18)
The compound 18 was prepared from 14 in 99% yield as the de-
scribed procedure for 16 as a white solid. 1H NMR (300 MHz, DMSO‑d6)
δ: 10.50 (s, 1H), 8.67–8.54 (m, 2H), 8.51 (d, J = 8.7 Hz, 2H), 7.95 (d,
J = 8.5 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 15.9 Hz, 1H),
7.43 (t, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 6.55 (d, J = 16.0 Hz,
1H), 4.19 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H), 1.25 (q, J = 7.0 Hz, 3H).
5.1.9. (E)-ethyl 3-(4-((4-chloro-6-(4-methoxyphenyl)-1,3,5-triazin-2-yl)
amino)phenyl)acrylate (14)
5.1.14. (E)-ethyl
3-(4-((4-(4-methoxyphenyl)-6-(4-(trifluoromethoxy)
Compound 13 (0.30 g, 0.88 mmol), 4-methoxyphenylboronic acid
(0.15 g, 0.97 mmol) and Pd(PPh3)2Cl2 (2 mg, 2.64 μmol) were sus-
pended in toluene (3 ml) and 2 M K2CO3 aqueous solution (0.24 g,
1.76 mmol). The suspension was stirred at 60 °C for 6 h under nitrogen
condition. After consumption of start material, the mixture was cooled
to room temperature, poured into water and extracted with di-
chloromethane. The organic layer was combined, washed with water
and brine and concentrated in vacuo to obtain crude product. The crude
product was purified by column chromatography (SiO2, PE:DCM = 2:1)
to afford compound 14 (0.27 g, 75%) as a white solid. 1H NMR
(300 MHz, CDCl3) δ: 8.42 (d, J = 8.4 Hz, 2H), 7.78–7.63 (m, 3H), 7.59
(d, J = 7.4 Hz, 3H), 7.01 (d, J = 8.7 Hz, 2H), 6.43 (d, J = 16.0 Hz,
1H), 4.30 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H).
phenyl)-1,3,5-triazin-2-yl)amino)phenyl)acrylate (19)
The compound 19 was prepared from 14 in 74% yield as the de-
scribed procedure for 16 as a white solid. 1H NMR (300 MHz, CDCl3) δ:
8.63 (d, J = 8.9 Hz, 2H), 8.56 (d, J = 8.9 Hz, 2H), 7.82 (d, J = 8.7 Hz,
2H), 7.71 (d, J = 15.8 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H),
7.37 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H), 6.43 (d,
J = 15.9 Hz, 1H), 4.29 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 1.36 (t,
J = 7.1 Hz, 3H).
5.1.15. (E)-3-(4-((4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)amino)
phenyl)acrylic acid (XHA1601)
Compound XHA1601 was prepared from 15 in 88% yield as the
described general procedure A as a pale yellow solid. mp: 238–240 °C.
1H NMR (300 MHz, DMSO‑d6) δ: 10.40 (s, 1H), 8.49 (d, J = 8.4 Hz,
4H), 7.95 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 7.8 Hz, 2H), 7.54 (d,
J = 15.9 Hz, 1H), 7.13 (d, J = 8.4 Hz, 4H), 6.47 (d, J = 15.7 Hz, 1H),
3.87 (s, 6H). 13C NMR (101 MHz, DMSO‑d6) δ: 170.11, 168.64, 164.18,
162.69, 142.75, 141.06, 130.50, 130.14, 128.72, 128.24, 120.05,
118.43, 113.96, 55.36. HRMS (ESI) m/z calcd for C26H22N4O4 [M+H]
+ 455.1720, found 455.1714.
5.1.10. (E)-ethyl
3-(4-((4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)
amino)phenyl)acrylate (15)
Compound 13 (0.70 g, 0.88 mmol), 4-methoxyphenylboronic acid
(0.69 g, 4.50 mmol) and Pd(PPh3)2Cl2 (4.20 mg, 6 μmol) were sus-
pended in toluene (7 ml) and 2 M K2CO3 aqueous solution (0.55 g,
4 mmol). The suspension was stirred at 100 °C for 6 h under nitrogen
condition. After completion of the reaction, the mixture was cooled to
room temperature, poured into water and extracted with di-
chloromethane. The organic layer was combined, washed with water
and brine and concentrated in vacuo to obtain crude product. The crude
product was purified by column chromatography (SiO2, PE:DCM = 2:1)
to afford compound 15 (0.70 g, 73%) as a white solid. 1H NMR
(300 MHz, DMSO‑d6) δ: 10.44 (s, 1H), 8.51 (d, J = 8.8 Hz, 4H), 7.97 (d,
J = 8.5 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 16.1 Hz, 1H),
7.14 (d, J = 8.9 Hz, 4H), 6.56 (d, J = 16.0 Hz, 1H), 4.19 (q,
J = 7.1 Hz, 2H), 3.88 (s, 6H), 1.26 (t, J = 7.1 Hz, 3H).
5.1.16. (E)-3-(4-((4-(4-methoxyphenyl)-6-phenyl-1,3,5-triazin-2-yl)
amino)phenyl)acrylic acid (XHA1602)
Compound XHA1602 was prepared from 16 in 97% yield as the
described general procedure A as a yellow solid. mp: 234–236 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 10.38(s, 1H), 8.41–8.47(m, 4H), 7.89(d,
J = 8.3 Hz,2H), 7.64(d, J = 8.3 Hz, 2H), 7.47–7.54(m, 4H), 7.05(d,
J = 8.6 Hz, 2H), 6.41(d, J = 16.0 Hz, 1H), 3.79(s, 3H). 13C NMR
(75 MHz, DMSO‑d6) δ: 171.04, 170.88, 168.89, 164.81, 163.32, 143.34,
141.43, 136.46, 132.80, 130.74, 129.37, 129.30, 129.15, 128.75,
128.58, 120.65, 118.85, 114.56, 55.91. HRMS (ESI) m/z calcd for
5.1.11. (E)-ethyl 3-(4-((4-(4-methoxyphenyl)-6-phenyl-1,3,5-triazin-2-yl)
amino)phenyl)acrylate (16)
C25H20N4O3 [M+H]+ 425.1614, found 425.1614.
Pd(PPh3)2Cl2 was added to a suspension of phenylboronic acid and
14 in toluene (5 ml) and 2 M K2CO3 (0.29 g, 2.10 mmol) aqueous so-
lution. The suspension was stirred at 100 °C for 6 h under nitrogen
condition. After completion of the reaction, the mixture was cooled to
room temperature and extracted with dichloromethane. And the or-
ganic layers were combined, washed with water and brine and con-
centrated in vacuum to obtain crude product. The crude product was
purified by column chromatography (SiO2, PE:DCM = 2:1) to afford 16
as a white solid (0.35 g, 73%). 1H NMR (300 MHz, DMSO‑d6) δ:
10.51(s, 1H), 8.51–8.56(m, 4H), 7.98(d, J = 8.6 Hz, 2H), 7.78(d,
J = 8.5 Hz, 2H), 7.59–7.67(m, 4H), 7.16(d, J = 8.7 Hz, 2H), 6.57(d,
J = 16.0 Hz, 1H), 4.19 (q, J = 7.0 Hz, 2H), 3.86(s, 3H), 1.27 (t,
J = 7.1 Hz, 3H).
5.1.17. (E)-3-(4-((4-(4-chlorophenyl)-6-(4-methoxyphenyl)-1,3,5-triazin-
2-yl)amino)phenyl)acrylic acid (XHA1603)
Compound XHA1603 was prepared from 17 in 59% yield as the
described general procedure A as a yellow solid. mp: > 280 °C. 1H NMR
(300 MHz, DMSO‑d6) δ: 10.26 (s, 1H), 8.36–8.32 (m, 4H), 7.80 (d,
J = 7.3 Hz, 2H), 7.62–7.44 (m, 4H), 6.99 (d, J = 7.9 Hz, 2H), 6.54 (d,
J = 15.6 Hz, 1H), 3.80 (s, 3H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.77,
170.67, 169.80, 164.53, 163.18, 140.55, 140.46, 137.42, 135.27,
130.63, 130.40, 130.30, 129.06, 128.56, 128.42, 123.61, 120.70,
114.33, 55.80. HRMS (ESI) m/z calcd for C25H19ClN4O3 [M+H]+
459.1225, found 459.1214.
5.1.18. (E)-3-(4-((4-(4-fluorophenyl)-6-(4-methoxyphenyl)-1,3,5-triazin-
2-yl)amino)phenyl)acrylic acid (XHA1604)
5.1.12. (E)-ethyl 3-(4-((4-(4-chlorophenyl)-6-(4-methoxyphenyl)-1,3,5-
triazin-2-yl)amino)phenyl)acrylate (17)
Compound XHA1608 was prepared from 18 in 57% yield as the
described general procedure A as a yellow solid. mp: 250–252 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 10.49 (s, 1H), 8.53 (t, J = 8.8 Hz, 2H),
8.52 (d, J = 8.5 Hz, 2H), 7.95 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.2 Hz,
2H), 7.54 (d, J = 16.2 Hz, 1H), 7.44 (t, J = 8.8 Hz, 2H), 7.15 (d,
J = 8.6 Hz, 2H), 6.48 (d, J = 16.1 Hz, 1H), 3.88 (s, 3H). 13C NMR
The compound 17 was prepared from 14 in 68% yield as the de-
scribed procedure for 16 as a white solid. 1H NMR (300 MHz, CDCl3) δ:
8.71–8.46 (m, 4H), 7.81 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 15.7 Hz, 1H),
7.60 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.41 (s, 1H), 7.04 (d,
9

X. Lu, et al.
BioorganicChemistry97(2020)103666
(75 MHz, DMSO‑d6) δ: 170.83, 170.00, 168.84, 166.92, 164.70, 163.61,
163.32, 143.01, 141.28, 132.94, 131.32, 131.21, 130.73, 129.49,
129.21, 128.49, 120.66, 119.26, 116.28, 115.99, 114.52, 55.88. HRMS
(ESI) m/z calcd for C25H19FN4O3 [M+H]+ 443.1520, found 443.1511
138.19, 130.37, 129.16, 128.14, 126.77, 120.29, 120.16, 115.43,
58.45, 35.92, 32.47. HRMS (ESI) m/z calcd for C27H25N5O4 [M + H]+
484.1986, found 484.1985.
5.1.24. (E)-3-(4-((4-(4-hydroxyphenyl)-6-phenyl-1,3,5-triazin-2-yl)
amino)phenyl)-N-(3-hydroxypropyl)acrylamide (XHA16108)
Compound XHA1610 was prepared from XHA1608 in 75% yield as
5.1.19. (E)-3-(4-((4-(4-methoxyphenyl)-6-(4-(trifluoromethoxy)phenyl)-
1,3,5-triazin-2-yl)amino)phenyl)acrylic acid (XHA1605)
Compound XHA1605 was prepared from 19 in 70% yield as the
described general procedure A as a yellow solid. mp: 248–250 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 10.53 (s, 1H), 8.64 (d, J = 8.6 Hz, 2H),
8.51 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 7.72 (d, J = 8.4 Hz,
2H), 7.65–7.43 (m, 3H), 7.15 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 16.0 Hz,
1H), 3.88 (s, 3H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.43, 169.30,
168.55, 164.20, 162.87, 151.12, 142.70, 140.74, 134.99, 130.37,
130.25, 128.99, 128.71, 127.90, 121.69, 120.71, 120.21, 118.28,
114.02, 55.38. HRMS (ESI) m/z calcd for C26H19F3N4O4 [M+H]+
509.1437, found 509.1437.
the described general procedure B as a white solid. mp: 240–242 °C. 1
H
NMR ((300 MHz, DMSO‑d6) δ: 10.36 (s, 1H), 10.25 (s, 1H), 8.46 (d,
J = 6.7 Hz, 2H), 8.36 (d, J = 8.3 Hz, 2H), 8.03 (s, 1H), 7.88 (d,
J = 8.1 Hz, 2H), 7.54 (s, 5H), 7.34 (d, J = 15.6 Hz, 1H), 6.89 (d,
J = 8.2 Hz, 2H), 6.51 (d, J = 15.3 Hz, 1H), 4.46 (t, J = 5.2 Hz, 1H),
3.38 (q, J = 5.8 Hz, 2H), 3.17 (q, J = 6.7 Hz, 2H), 1.55 (p, J = 6.5 Hz,
2H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.30, 165.23, 164.31, 161.66,
161.61, 140.37, 138.12, 136.04, 132.25, 130.50, 129.36, 128.66,
128.23, 128.15, 126.50, 120.51, 120.29, 115.51, 58.42, 35.90, 32.47.
HRMS (ESI) m/z calcd for C27H25N5O3 [M + H]+ 468.2036, found
468.2034.
5.1.20. (E)-3-(4-((4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)amino)
phenyl)-N-(3-hydroxypropyl)acrylamide (XHA1606)
5.1.25. 2,4-dichloro-6-(4-methoxyphenyl)-1,3,5-triazine (25)
Compound XHA1606 was prepared from XHA1601 in 59% yield as
Compound 12 (2 g, 10.85 mmol), 4-methoxyphenylboronic acid
(1.10 g, 7.23 mmol) and Pd(PPh3)2Cl2 (25 mg, 36.15 μmol) were sus-
pended in toluene (40 ml) and 2 M K2CO3 aqueous solution (4 g,
28.92 mmol). The suspension was stirred at 60 °C for 12 h under ni-
trogen condition. After completion of the reaction, the mixture was
cooled to room temperature, concentrated and extracted with di-
chloromethane. The organic layer was combined, washed with water
and brine and concentrated in vacuo to obtain crude product. The crude
product was purified by column chromatography (SiO2, PE:DCM = 5:1)
to afford compound 25 (0.81 g, 44%) as a white solid. 1H NMR
(300 MHz, CDCl3) δ: 8.46 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H),
3.93 (s, 3H).
the described general procedure B as a white solid. mp: 230–232 °C. 1
H
NMR (300 MHz, DMSO‑d6) δ: 10.34 (s, 1H), 8.48 (d, J = 8.0 Hz, 4H),
8.07 (t, J = 5.7 Hz, 1H), 7.94 (d, J = 7.7 Hz, 2H), 7.60 (d, J = 7.4 Hz,
2H), 7.41 (d, J = 15.1 Hz, 1H), 7.12 (d, J = 8.1 Hz, 4H), 6.57 (d,
J = 15.7 Hz, 1H), 4.52 (t, J = 5.2 Hz, 1H), 3.86 (s, 6H), 3.45 (q, 6.0 Hz,
2H), 3.25 (q, 6.7 Hz, 2H), 1.62 (p, J = 6.5 Hz, 2H). 13C NMR (75 MHz,
DMSO‑d6) δ: 170.64, 165.75, 164.70, 163.21, 140.89, 138.64, 130.67,
129.58, 128.74, 128.67,120.96, 120.75, 114.53, 58.91, 55.90, 36.40,
32.97. HRMS (ESI) m/z calcd for C29H29N5O4 [M+H]+ 512.2299,
found 512.2301.
5.1.21. (E)-3-(4-((4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)amino)
phenyl)acrylic acid (XHA1607)
5.1.26. 2-chloro-4,6-bis(4-methoxyphenyl)-1,3,5-triazine (26)
Compound 25 (0.81 g, 3.16 mmol), 4-methoxyphenylboronic acid
(0.53 g, 3.48 mmol) and Pd(PPh3)2Cl2 (7 mg, 10.08 μmol) were sus-
pended in toluene (9 ml) and 2 M K2CO3 aqueous solution (0.87 g,
6.72 mmol). The suspension was stirred at 60 °C for 6 h under nitrogen
condition. After completion of the reaction, the mixture was cooled to
room temperature, concentrated and extracted with dichloromethane.
The organic layer was combined, washed with water and brine and
concentrated in vacuo to obtain crude product. The crude product was
purified by column chromatography (SiO2, PE:DCM = 5:1) to afford
compound 26 (0.87 g, 67%) as a white solid. 1H NMR (300 MHz,
CDCl3) δ: 8.56 (d, J = 8.4 Hz, 4H), 7.04 (d, J = 8.4 Hz, 4H), 3.93 (s,
6H).
Compound XHA1607 was prepared from XHA1601 in 70% yield as
the described general procedure C as a white solid. mp: 274–276 °C. 1
H
NMR (300 MHz, DMSO‑d6) δ: 10.31 (s, 2H), 8.42 (d, J = 7.9 Hz, 4H),
7.98 (d, J = 7.6 Hz, 2H), 7.74 (d, J = 7.9 Hz, 2H), 7.58 (d,
J = 15.8 Hz, 1H), 6.96 (d, J = 7.9 Hz, 4H), 6.47 (d, J = 15.6 Hz, 1H).
13C NMR (75 MHz, DMSO‑d6) δ: 170.23, 167.78, 164.21, 161.48,
143.67, 141.42, 130.37, 129.00, 128.03, 126.69, 119.91, 117.07,
115.43. HRMS (ESI) m/z calcd for C24H18N4O4 [M+H]+ 427.1407,
found 427.1401.
5.1.22. (E)-3-(4-((4-(4-hydroxyphenyl)-6-phenyl-1,3,5-triazin-2-yl)
amino)phenyl)acrylic acid (XHA1608)
Compound XHA1608 was prepared from XHA1602 in 80% yield as
the described general procedure C as a yellow solid. mp: 274–276 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 10.40 (s, 1H), 8.46 (d, J = 6.9 Hz, 2H),
8.36 (d, J = 7.3 Hz, 2H), 7.90 (d, J = 6.4 Hz, 2H), 7.67 (d, J = 6.9 Hz,
2H), 7.61–7.29 (m, 4H), 6.89 (d, J = 6.2 Hz, 2H), 6.40 (d, J = 15.0 Hz,
1H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.03, 167.69, 165.69, 163.98,
161.71, 143.27, 141.15, 135.54, 132.11, 130.53, 128.97, 128.69,
128.62, 128.23, 126.44, 120.06, 117.42, 115,53. HRMS (ESI) m/z calcd
for C24H18N4O3 [M + H]+ 411.1458, found 411.1458.
5.1.27. 4-(4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)benzaldehyde
(27)
Compound 26 (0.80 g, 2.41 mmol), 4-formylphenylboronic acid
(0.40 g, 3.48 mmol) and Pd(PPh3)2Cl2 (15 mg, 24.10 μmol) were sus-
pended in DMF (9 ml) and 2 M K2CO3 aqueous solution (0.67 g,
4.82 mmol). The suspension was stirred at 120 °C for 6 h under nitrogen
condition. After completion of the reaction, the mixture was cooled to
room temperature, poured into ice water and extracted with ethyl
acetate. The organic layer was combined, washed with water and brine
and concentrated in vacuo to obtain crude product. The crude product
was purified by column chromatography (SiO2, PE:DCM = 2:1) to af-
ford compound 27 (0.50 g, 74%) as a white solid. 1H NMR (300 MHz,
DMSO‑d6) δ 10.11 (s, 1H), 8.76 (d, J = 8.2 Hz, 2H), 8.57 (d,
J = 8.8 Hz, 4H), 8.07 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 8.9 Hz, 4H),
3.86 (s, 6H).
5.1.23. (E)-3-(4-((4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)amino)
phenyl)-N-(3-hydroxypropyl)acrylamide (XHA1609)
Compound XHA1609 was prepared from XHA1607 in 75% yield as
the described general procedure B as a yellow solid. mp: 270–272 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 10.27 (s, 1H), 10.25 (s, 2H), 8.42 (d,
J = 8.8 Hz, 4H), 8.08 (t, J = 5.7 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H),
7.62 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 15.7 Hz, 1H), 6.97 (d,
J = 8.8 Hz, 4H), 6.59 (d, J = 15.7 Hz, 1H), 4.52 (t, J = 5.2 Hz, 1H),
5.1.28. (E)-ethyl
3-(4-(4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)
347 (q, 5.7 Hz, 2H), 3.26 (q, 6.7 Hz, 2H), 1.64 (p, J = 6.5 Hz, 2H). 13
C
phenyl)acrylate(28)
NMR (101 MHz, DMSO‑d6) δ: 170.24, 165.29, 164.22, 161.46, 140.59,
Ethyl 2-tri(phenyl)phosphoranylideneacetate (0.57 g, 1.64 mmol)
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X. Lu, et al.
BioorganicChemistry97(2020)103666
was added to a solution of 27 in dried dichloromethane (13 ml). The
mixture was stirred at room temperature for 16 h under nitrogen con-
dition. After completion of the reaction, the mixture was concentrated
in vacuum and was purified by column chromatography (SiO2,
PE:DCM = 2:1) to afford compound 28 (0.50 g,85%). 1H NMR
(300 MHz, CDCl3) δ: 8.78–8.72 (m, 6H), 7.80 (d, J = 16.1 Hz, 1H), 7.72
(d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.8 Hz, 4H), 6.59 (d, J = 15.9 Hz,
1H), 4.32 (q, J = 7.2 Hz, 2H), 3.95 (s, 6H), 1.39 (t, J = 7.1 Hz, 3H).
J = 15.8 Hz, 1H), 4.80 (s, 1H), 3.49 (t, J = 5.9 Hz, 2H), 3.30 (t,
J = 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6) δ: 170.91, 170.20,
165.35, 162.63, 139.46, 138.08, 136.97, 131.27, 129.45, 128.31,
126.70, 124.85, 116.19, 60.40, 42.27. HRMS (ESI) m/z calcd for
C
26H22N4O4 [M + H]+ 455.1720, found 455.1722.
5.1.34. (E)-3-(4-(4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)phenyl)-N-
(3,3,3-trifluoropropyl)acrylamide (XHA1616)
Compound XHA1616 was prepared from XHA1613 in 58% yield as
the described general procedure B as a white solid. mp: > 280 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 10.35 (s, 2H), 8.89 (t, J = 6.4 Hz, 1H),
8.72 (d, J = 8.5 Hz, 2H), 8.59 (d, J = 8.8 Hz, 4H), 7.84 (d, J = 8.3 Hz,
2H), 7.65 (d, J = 15.8 Hz, 1H), 6.99 (d, J = 8.8 Hz, 4H), 6.87 (d,
J = 15.9 Hz, 1H), 4.14–4.02 (m, 2H). 13C NMR (75 MHz, DMSO‑d6) δ:
170.88, 170.12, 165.83, 162.53, 139.98, 138.94, 137.31, 131.29,
129.47, 128.59, 126.70, 123.92, 123.15, 116.16. HRMS (ESI) m/z calcd
for C26H19F3N4O3 [M + H]+ 493.1488, found 493.1488.
5.1.29. (E)-3-(4-(4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)phenyl)
acrylic acid (XHA1611)
Compound XHA1611 was prepared from 28 in 70% yield as the
described general procedure A as a yellow solid. mp: 238–240 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 8.66–8.61 (m, 6H), 7.88 (d, J = 8.2 Hz,
2H), 7.65 (d, J = 16.4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 4H), 6.69 (d,
J = 16.0 Hz, 1H), 3.87 (s, 6H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.59,
170.06, 168.15, 163.51, 142.72, 138.74, 137.18, 130.90, 129.18,
128.75, 128.14, 122.41, 114.52, 55.84. HRMS (ESI) m/z calcd for
C
26H21N3O4 [M + H]+ 440.1611, found 440.1606.
5.2. Biological evaluation
5.1.30. (E)-3-(4-(4,6-bis(4-methoxyphenyl)-1,3,5-triazin-2-yl)phenyl)-N-
(3-hydroxypropyl)acrylamide (XHA1612)
5.2.1. Cell culture
MCF-7 and Ishikawa cells were cultured in Dulbecco's Modified
Eagle Medium (DMEM, KeyGEN Biotech),containing 10% fetal bovine
serum (FBS, GIBCO), 100U/ml Penicillin and 100 mg/ml Streptomycin,
at 37 °C under 5% CO2 condition in humidified atmosphere.
Compound XHA1612 was prepared from XHA1611 in 67% yield as
the described general procedure A as a white solid. mp: 230–232 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 8.91–8.39 (m, 6H), 8.18 (t, J = 5.3 Hz,
1H), 7.71 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 15.7 Hz, 1H), 7.07 (d,
J = 8.3 Hz, 4H), 6.74 (d, J = 15.8 Hz, 1H), 4.47 (t, J = 5.1 Hz, 1H),
3.83 (s, 6H), 3.43 (q, J = 5.7 Hz, 2H), 3.23 (q, J = 6.3 Hz, 2H), 1.61 (p,
J = 6.5 Hz, 2H). 13C NMR (75 MHz, DMSO‑d6) δ: 170.22, 169.79,
164.69, 163.12, 138.99, 137.49, 136.17, 130.51, 128.93, 127.79,
127.70, 124.23, 114.15, 58.42, 55.43, 36.00, 32.39. HRMS (ESI) m/z
calcd for C29H28N4O4 [M + H]+ 497.2190, found 497.2183.
5.2.2. MTT assay
Trypsinized-cells (100 μl, 5 × 104 cells/ml) were plated in the wells
of 96-well plates and incubated for 24 h. Then, 100 μl medium which
contained the test compounds at five different doses range of 1.6 × 10-4
mol/L, 8 × 10-5 mol/L, 4 × 10-5 mol/L, 2 × 10-5 mol/L and 2 × 10-6
mol/L was added to each well, and these plates were re-incubated for
additional 48 h. Equivalent volume medium including 0.1% DMSO was
set as control. After that, 20 μl MTT (5 mg/ml) was added to each well
in darkness and incubated for 4 h. Next, the culture medium was dis-
carded and 150 μl DMSO was added instead and the plates were then
placed at shaker for 10 min to dissolve the purple formazan crystals
adequately. The absorbance values were measured at 490 nm and fi-
nally GraphPad Prism 7.0 was utilized to calculate the IC50 value of
each compounds.
5.1.31. (E)-3-(4-(4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)phenyl)
acrylic acid (XHA1613)
Compound XHA1613 was prepared from XHA1611 in 67% yield as
the described general procedure C as a yellow solid. mp: > 280 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 12.53 (s, 1H), 10.33 (s, 2H), 8.67 (d,
J = 7.9 Hz, 2H), 8.57 (d, J = 8.1 Hz, 4H), 7.91 (d, J = 7.6 Hz, 2H),
7.71 (d, J = 15.9 Hz, 1H), 6.99 (d, J = 8.2 Hz, 4H), 6.70 (d,
J = 15.9 Hz, 1H). 13C NMR(75 MHz, DMSO‑d6) δ: 170.38, 169.26,
167.37, 162.00 142.63, 137.91, 137.12, 130.79, 128.80, 128.52,
5.2.3. Real-Time polymerase chain reaction (RT-PCR)
126.19, 121.16, 115.65. HRMS (ESI) m/z calcd for
C
24H17N3O4
RNA samples were reverse transcribed to cDNA and the PCR reac-
tions were performed using TaKaRa SYBR Green Master Mix (Code. no.
638320) carried out in StepOnePlusTM Real-Time PCR instrument
(4376600, Life Technologies). The program for amplification was 1
cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60 °C for
30 s, and 95 °C for 10 s. The PCR results were normalized to GAPDH
expression and were quantified by the ΔΔCT method.
[M + H]+ 412.1298, found 412.1295.
5.1.32. (E)-3-(4-(4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)phenyl)-N-
(3-hydroxypropyl)acrylamide (XHA1614)
Compound XHA1614 was prepared from XHA1613 in 60% yield as
the described general procedure B as a white solid. mp: > 280 °C. 1H
NMR (300 MHz, DMSO‑d6) δ: 10.34 (s, 2H), 8.69 (d, J = 7.6 Hz, 2H),
8.58 (d, J = 8.1 Hz, 4H), 8.22 (t, J = 5.7 Hz, 1H), 7.80 (d, J = 7.6 Hz,
2H), 7.54 (d, J = 15.8 Hz, 1H), 7.00 (d, J = 8.1 Hz, 4H), 6.81 (d,
J = 15.9 Hz, 1H), 4.50 (t, J = 5.2 Hz, 1H), 3.48 (q, J = 5.9 Hz, 2H),
3.27 (q, J = 6.5 Hz, 2H), 1.66 (p, J = 6.2 Hz, 2H). 13C NMR (75 MHz,
DMSO‑d6) δ: 170.38, 169.39, 164.69, 162.01, 138.96, 137.42, 136.37,
130.77, 128.96, 127.82, 126.23, 124.16, 115.66, 58.41, 35.99, 32.39.
HRMS (ESI) m/z calcd for C27H24N4O4 [M + H]+ 469.1877, found
469.1875.
5.2.4. Western blotting
MCF-7 were cultured according to the literature with a little mod-
ification [29]. MCF-7 were cultured in six-well plate (105 cells/well) at
four concentrations (10-9–10-6 M) for 5 days. Then,Cells with different
treatments were washed twice with PBS, then collected and lysed in
lysis buffer (Servicebio, NO. G2002) for 1 h on the ice. The lysates were
then subjected to centrifugation (13,000 rpm) at 4 °C for 20 min. Pro-
tein concentration in the supernatants was detected by BCA protein
assay (Thermo, Waltham, MA). And the protein concentration was di-
luted with protein loading buffer at the radio of protein concentration:
protein loading buffer (4:1). Then, equal amount of protein was sepa-
rated with 12% SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes (Millipore, Bedford, MA) using a semi-dry transfer
system (Bio-rad, Hercules, CA). Proteins were detected using specific
antibodies overnight at 4 °C and then were washed with TBST three
times and five minutes every time. Next, the proteins were added with
5.1.33. (E)-3-(4-(4,6-bis(4-hydroxyphenyl)-1,3,5-triazin-2-yl)phenyl)-N-
(2-hydroxyethyl)acrylamide (XHA1615)
Compound XHA1615 was prepared from XHA1613 in 78% yield as
the described general procedure B as a red solid. mp: 248–250 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 10.41 (s, 2H), 8.71 (d, J = 8.4 Hz, 2H),
8.59 (d, J = 8.8 Hz, 4H), 8.29 (t, J = 5.7 Hz, 1H), 7.81 (d, J = 8.4 Hz,
2H), 7.55 (d, J = 15.7 Hz, 1H), 7.00 (d, J = 8.8 Hz, 4H), 6.87 (d,
11

X. Lu, et al.
BioorganicChemistry97(2020)103666
secondary antibodies and incubated for 30 min at room temperature.
After that, proteins were washed with TBST three times and five min-
utes every time. Finally, the PVDF membranes were reacted with pre-
prepared ECL reaction solution for 1–2 min and the visualized bands
were captured by film which was optimized of the bands color by
Photoshop and analyzed by Alpha software for the optical density value
of the target bonds.
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performed step by step. Four thousand steps of steepest descent method
were firstly employed and then followed by six thousand steps of con-
jugate gradient method for energy minimization. Then the system was
heated under canonical ensemble from 0 to 303 K in 300 ps with the
Langevin thermostat applied, the force constant for the harmonic re-
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equilibrated for 10 ns under NPT conditions (with constant pressure
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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Acknowledgements
This work was supported by grants from the Natural Science
Foundation of China (NSFC,81874286) and We thank Shenzhen Shuli
Tech Co., Ltd for technical support for the MD simulations in this work.
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Appendix A. Supplementary material
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