SARs of a novel series of s-triazine compounds targeting vimentin to induce methuotic phenotype

Article abstract of DOI:10.1016/j.ejmech.2021.113188

Herein, we describe the design, synthesis and structure?activity relationships of a series of novel s-triazine compounds can induce methuotic phenotype in various types of cancer cells. (E)-1-(4-Chlorophenyl)-3-(4-((4-morpholino-6-styryl-1,3,5-triazine-2-yl)amino)phenyl)urea, compound V6, exhibited a striking methuotic phenotype with a minimal effective concentration of less than 10 nM in U87 glioblastoma cells. Based on structure?activity relationship studies, we designed and synthesized an active probe P1 that retained the full potential of V6 in inducing the methuotic phenotype in U87 glioblastoma cells. Using this probe following affinity-based proteomic profiling strategy, we identified vimentin as the specific target protein of compound V6. Molecular docking revealed that V6 can form hydrogen bonds with vimentin at ²⁷³R and ²⁷⁶Y in its rod domain.

Full text of DOI:10.1016/j.ejmech.2021.113188

European Journal of Medicinal Chemistry 214 (2021) 113188
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
SARs of a novel series of s-triazine compounds targeting vimentin to
induce methuotic phenotype
,
,
d
,
, e,
Lei Zhang a 1, Zhipeng Qu b 1, Jianping Wu d 1, Shining Yao c, Qingqing Zhang a,
Tao Zhang b, Lian Mo f, Qizheng Yao a *, Ying Xu b **, Ruihuan Chen b
,
,
, d, f, ***
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, Jiangsu, 210009, China
b Cambridge-Suda Genomic Resource Center, Medical College of Soochow University, Suzhou, Jiangsu, 215123, China
c Najing Shiqi Pharmaceutical Co. Ltd., Nanjing, Jiangsu, 211198, China
d Luoda Biosciences, Inc., Chuzhou, Auhui, 239234, China
e Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210046, China
f Aluda Pharmaceuticals, Inc., Union City, CA, 94587, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe the design, synthesis and structureꢀactivity relationships of a series of novel s-
triazine compounds can induce methuotic phenotype in various types of cancer cells. (E)-1-(4-
Chlorophenyl)-3-(4-((4-morpholino-6-styryl-1,3,5-triazine-2-yl)amino)phenyl)urea, compound V6,
exhibited a striking methuotic phenotype with a minimal effective concentration of less than 10 nM in
U87 glioblastoma cells. Based on structureꢀactivity relationship studies, we designed and synthesized an
active probe P1 that retained the full potential of V6 in inducing the methuotic phenotype in U87
glioblastoma cells. Using this probe following affinity-based proteomic profiling strategy, we identified
vimentin as the specific target protein of compound V6. Molecular docking revealed that V6 can form
hydrogen bonds with vimentin at 273R and 276Y in its rod domain.
Received 8 November 2020
Received in revised form
5 January 2021
Accepted 7 January 2021
Available online 18 January 2021
Keywords:
Methuosis
s-Triazine derivatives
Vimentin
© 2021 Elsevier Masson SAS. All rights reserved.
Target
Cancer
1. Introduction
Vimentin is a highly conserved type-III intermediate filament
(IF) protein. It is expressed in abundance in cells of mesenchymal
Nonapoptotic forms of cell death are now recognized as playing
significant roles during embryonic development, neuro-
degeneration, and cancer regression [1]. In these cases, loss of cell
viability may occur in a manner that is independent of caspase
activation. Methuosis is a novel form of nonapoptotic cell death. Its
diagnostic morphologic feature is the massive accumulation of
vacuoles derived from macropinosomes, ultimately causing cells to
detach from the substratum and rupture [2]. Methuosis has been
reported to occur in glioblastoma, where it can be induced by a
chalcone-like compound. Nevertheless, the targets of the
methuosis-inducing chalcone were still unclear.
origin, and also dynamically in various types of cells during
different differentiation stages. Vimentin IF forms dynamic and
flexible cytoskeletal networks that dictate the structural and me-
chanical properties of the cell and its organelles [6]. It induces
changes in cell shape, motility, and adhesion during the epithelial-
to-mesenchymal transition (EMT) [7,8]. associates with motor
proteins and plays an important role in membrane trafficking and
endolysosomal vesicle transportation [9e13]. Therefore, the
involvement of vimentin in cytoplasmic vacuolization and
methuosis appears to be logically expected.
A common approach for identifying drug targets is photoaffinity
labeling (PAL). PAL is a powerful technique in chemical biology and
chemical proteomics, and has been successfully demonstrated on a
variety of non-covalent proteinedrug interactions under native
cellular conditions [3e5]. To make the corresponding probe, the
drug is typically derivatized with a photo-reactive group and a
reporter group (referred to herein as the linker). One of the most
essential considerations in PAL is that the probe must retain most, if
not all, of the original biological activity of the drug after
* Corresponding author.
** Corresponding author.
*** Corresponding author. Cambridge-Suda Genomic Resource Center, Medical
College of Soochow University, Suzhou, Jiangsu, 215123, China.
E-mail addresses: alpszhanglei@163.com (L. Zhang), qz_yao@163.com (Q. Yao),
yingxu@suda.edu.cn (Y. Xu), ruihuan@aludapharm.com (R. Chen).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113188
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
derivatization with the linker. To achieve this, a nonessential site in
the probe must be found which can be replaced with a linker
without compromising the target-binding properties. Unfortu-
nately, however, for chalcone-like compounds, no nonessential
sites that could be used to linked to a photo-reactive group and a
reporter group without loss of capacity to induce methuosis have
been identified [2].
Herein, we describe the design, synthesis and structure-activity
relationships of a series of novel s-triazine compounds, which can
induce methuotic phenotype in various types of cancer cells. After
investigating the tolerance to substitution of rings A and C of the s-
triazines, the 4-position of ring C was identified as a nonessential
site that could attach a linker to produce the probe. Based on
structureꢀactivity relationships studies, we designed and synthe-
sized an active probe that retained the full potential of the s-
triazine compounds in inducing the methuotic phenotype in glio-
blastoma cells. Using this probe and the PAL strategy, we success-
fully identified vimentin as the target of the s-triazine compounds.
impact on activity measured by dose response of viability (ATP) of
GCs determined IC50 (24 h). bThe inducing vacuolization ability was
scored. The scoring standard is: the score is 5 if the compound
caused a significant vacuole when the compound concentration
was 10 nm, and the order was: 31.6 nm, score 4; 100 nm, score 3;
316 nm, score 2; 1000 nm, score 1; without no vacuole at any
concentrations, score 0. cV12 as a key intermediate to prepare
V13eV20, the growth inhibitory effects and inducing the
morphological vacuolization ability was not detected.
To investigate the impact of the phenylenediamine substituent
on biological activity, the para-phenylenediamine substituent at
the 2-position on the triazine ring was replaced with a meta-phe-
nylenediamine substitution in compounds V21eV23 (Table 2). All
meta-substituted analogues, V21eV23, were ineffective at inducing
vacuolization, indicating that the para-phenylenediamine substit-
uent at the 2-position of the triazine is a key feature required for
activity.
2.2. Chemistry
2. Results and discussion
The para-phenylenediamine substituted s-triazine derivatives
V1eV12 were synthesized in eight steps as shown in Scheme 1.
Compound 1 was prepared by methylation of 2,4,6-trichloro-1,3,5-
triazine with methylmagnesium bromide. Then 1 was reacted with
various substituted benzaldehydes in the presence of concentrated
aqueous HCl to afford 2a-d in moderate yields. Intermediates 2a-
d were chlorinated with phosphorus oxychloride to form 3a-d.
Compound 4 was prepared by Boc-protection of benzene-1,4-
diamine and was then coupled with various substituted iso-
cyanatobenzenes to afford ureas 5a-d in moderate yields. Boc-
deprotection of 5a-d in the presence of trifluoroacetic acid pro-
vided amines 6a-d which upon heteroarylation with 3a-d in the
presence of DIPEA generated the key intermediates 7a-n, which
were subsequently aminated with various amines to afford com-
pounds V1eV12 in yields of 56e78%.
As shown in Scheme 2, the amide s-triazine derivatives
V13eV20 were synthesized in two steps. Initially, the key inter-
mediate ester V12 was hydrolyzed with LiOH monohydrate to
afford carboxylic acid 8, which was subsequently acylated with
various substituted amines using N-ethylcarbodiimide (EDCI), 1-
hydroxybenzotriazole (HOBt), and triethylamine to afford the
target amide s-triazine derivatives V13eV20 in 59e76% yield.
The meta-phenylenediamine substituted s-triazine analogues
V21eV23 were prepared in five-step as described in Scheme 3.
Compound 9 was prepared by Boc-protection of benzene-1,3-
diamine and the product was then reacted with 1-chloro-4-
isocyanatobenzene to afford urea 10. Boc-deprotection of 10 in
the presence of trifluoroacetic acid produced amine 11 which upon
reaction with dichlorotriazines 3a-c in the presence of DIPEA
generated key intermediates 12a-c. Finally, reaction with mor-
pholine afforded the meta-phenylenediamine substituted s-
triazine derivatives V21eV23 in 66e82% yield.
2.1. Structure activity relationships (SARs)
In searching for effective anti-cancer therapeutics, we discov-
ered s-triazine derivative V1 with a morpholine substituent in the
4-position of the triazine ring. Owing to the observation that this
compound had the ability to induce massive vacuolization in
various types of cancer cells (Fig.1), we initiated a SAR study around
V1.
The SAR study of the triazine ring substitution is shown in
Table 1. First, to investigate the importance of the morpholine
group in the 4-position of the triazine, multiple analogues with 1-
methyl-4-piperazine, dimethylamine, pyrrolidine or 3-hydroxyl
azetidine groups (V2eV5) were synthesized and evaluated for
their abilities to induce intracellular vacuolization. It was found
that the morpholine group was essential to induce intracellular
vacuolation of tumor cells; replacement of the morpholine sub-
stituent (V1) with other substituents (V2eV5) rendered the com-
pounds inactive (Table 1).
To find nonessential sites of the s-triazine derivatives for the
design of a probe compound, the flexibility and tolerance of sub-
stituents on rings A and C were investigated. Since the morpholine
substituent at the 4-position was critical for activity, an initial set of
analogues (V6eV11) was designed and synthesized with different
modifications of the ring A, while retaining the morpholine sub-
stituent at the 4-position (Table 1). Intriguingly, all the compounds
retained an ability to induce vacuolization in tumor cells, albeit
with slight differences in potency. Among them, compound V6 (R1
is H) showed the strongest effect with massive vacuoles being
accumulated inside the cells treated with the compound in a dose-
dependent manner, with a minimal effective concentration of less
than 10 nM. It appeared that compounds with smaller R1 groups,
such as V6 and V7 with R1 being H, had stronger effects than those
compounds with larger R1 substituents (V8eV11), suggesting that
there was little flexibility for substituents on ring A to maintain
activity. To further investigate the flexibility and tolerance of the 4-
position of ring C, a series of amide analogues (V13eV20) of V6 that
possessed an amide moiety in the 4-position of the ring C were
synthesized and their effects on inducing morphological vacuoli-
zation were explored. Gratifyingly, all newly synthesized amide
compounds induced vacuolization in tumor cells, which suggested
large flexibility for substituents on ring C (Table 1). This flexibility
enabled us to label compound V6 for a target deconvolution study.
aRepresentative compounds (compound ID) with various com-
binations of structural modification at sites R1,R2 and R3 and their
2.3. Methuotic phenotype
The key characteristic feature of the methuotic process is
intracellular vacuolization caused by disturbed micropinocytosis.
To confirm that the compound-induced vacuolization was the
result of an altered macropinocytotic process, and owing to its
potent activity, compound V6 was chosen for further studies. In
U87 cells, at concentrations of as low as 10 nM, V6 resulted in a
clear accumulation of intracellular vacuoles (Fig. 2a) in the para-
nuclear region that became readily visible in less than 2 h after
treatment (Fig. 2b). This rapid effect suggests direct involvement of
V6 target protein(s) in the cytoplasmic vacuolization. Lucifer Yellow
2

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Fig. 1. Inducing the morphological vacuolization of various cancer cells using V1 at a concentration of 100 nM.
(LY) assay [14] and staining for ER and Golgi markers confirmed
that the vacuoles originated solely from macropinocytosis (Fig. 2c),
which is characteristic of methuosis, consistent with previous re-
ports [15e17]. Time-lapse confocal microscopy clearly revealed that
small vesicles moved rapidly and continuously from near the
cytoplasmic membrane at the far ends toward the center of the
cells, then fused with each other to form large vacuoles (Fig. 2c,
upper panel). With increasing number and size of the cytoplasmic
vacuoles, the cells, especially the newly-divided ones gradually
became rounded and eventually disintegrated (Fig. 2c, lower
panel). These phenotypic alterations indicated that V6 not only
stimulated the formation of macropinosomes but also impeded
their further degradation, thereby mediating a typical methuosis
event.
therefore unlikely to alter the binding ability of the compound to its
target protein. The linker L2 [21], which has been successfully used
for target engagement studies with other drugs (eg. Imatinib, Sor-
afeinib), consisted of a photosensitive group (3H-diazirin-3-yl),
which minimizes interference with protein binding owing to its
small size and the need of a short irradiation time after UV-A
exposure to generate the highly reactive carbene species. The
linker also contained a terminal alkyne handle, which would be
used for subsequent ex vivo pull-down/target identification by
conjugation to a suitable reporter molecule (biotin-N3; boxed in
Fig. 3b) via bio-orthogonal click chemistry. The synthesis of the
probe P1 is showed in Scheme 4. Key intermediate carboxylic acid 8
was coupled with the amine precursor L2 of the linker, which was
obtained following a previously reported procedure [21], using
EDCI, HOBt and triethylamine to afford the target probe P1.
The click reporter (S1) [22] was synthesized by linking biotin,
which can be used for protein detection and isolation, to a long
chain-linked azido-group which can react with the ethynyl group of
the probe molecule in situ by a click reaction (Fig. 3b) to form a
triazole.
2.4. Photoaffinity labeling
In order to identify target proteins that were responsible for the
chemical-induced methuosis, an affinity-based proteomic profiling
strategy [18e20] was used. The study required a specific probe and
a corresponding click reporter. The probe molecule (P1, Fig. 3a) was
derived from compound V6 by replacing the chloro group at the 4-
position of ring C with a linker. This position was shown in the SAR
studies to have minimal or no impact on the activity and was
First, it was necessary to confirm that the probe P1 maintained
the same vacuolizating-inducing activity as V6 in U87 cells. As ex-
pected, the probe molecule P1 was cell-permeable and able to
induce the same phenotype as V6 did with comparable strength
3

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Table 1
Effects of s-triazine analogues on the growth inhibition and vacuolization induction in cultured U87 GBM cells.
compound
R1
R2
R3
IC50
(
m
M)a
Vacuole formation
yes
Vacuolization scoreb
4
V1
3-NO2
4-Cl
1.71 0.06
V2
3-NO2
4-Cl
1.21 0.07
no
0
V3
V4
3-NO2
3-NO2
4-Cl
4-Cl
5.12 0.28
4.47 0.39
no
no
0
0
V5
3-NO2
4-Cl
1.32 0.22
no
0
V6
H
4-Cl
4-F
4-F
4-F
3-F
4- F
4.79 0.55
4.75 0.87
1.33 0.13
2.50 0.08
4.41 0.43
1.11 0.10
/
yes
yes
yes
yes
yes
yes
/
5
V7
H
4.5
4
V8
3-Cl
4-Cl
4-Cl
3-NO2
H
V9
2
V10
V11
V12c
2
3.5
/
V13
V14
H
H
0.53 0.02
0.87 0.08
yes
yes
3.5
1
V15
V16
H
H
0.23 0.01
2.48 0.44
yes
yes
2.5
4.5
V17
V18
V19
V20
H
H
H
H
2.31 0.29
1.43 0.10
1.11 0.06
0.55 0.05
yes
yes
yes
yes
3.5
1
1
3.5
and speed (Fig. 4), indicating that the probe could indeed effectively
minimize the influence of the linker on target protein binding or
biological activity.
of 55KD and 220KD were detected and successfully isolated
(Fig. 5b). Protein sequencing showed that the two proteins shared
an identical sequence, matching with that of the intermediate
filament protein, vimentin (Fig. 5c). The 55KD protein appeared to
be a vimentin monomer while the 220KD protein was a vimentin
tetramer.
With no previous reports showing direct connections between
vimentin and methuosis, we wanted to further characterize this
finding. To explore the domain of vimentin that the compound
binds to, compound V6 was docked in silico with vimentin utilizing
the C-DOCKER program within Discovery Studio 3.0 software
In vitro labeling for the potential probe-target protein(s) using
probe P1 was conducted in U87 cells. Live U87 cells were treated
with the probe P1 at 100 nM for 2 h followed by UV irradiation to
initiate photo-crosslinking as covalent capture and a click reaction
between the terminal alkyne and the azido group of biotin-N3 (S1).
Enrichment using avidin-agarose beads, separation by SDS-PAGE,
and LC-MS/MS analysis (the labeling process is shown in Fig. 5a),
two specific P1 (V6 core)-binding proteins with molecular weights
4

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Table 2
3. Conclusion
Effects of meta-phenylenediamine substituted analogues on the growth inhibition
and vacuolization induction in cultured U87 GBM cells.
In summary, a series of novel s-triazine compounds were
designed, synthesized and evaluated for their biological activities
by phenotypic changes of cancer cells. The functional groups critical
for the activity of the compounds were defined from the SAR
studies. The target protein for this type of vacuolization-inducing
compound was identified as vimentin intermediate filament pro-
tein through direct photoaffinity labeling (PAL) and subsequent
protein sequencing. The potential binding sites were located at the
rod domain of the vimentin protein where 273R and 276Y form
hydrogen bonding with the nitrogen atoms of the s-triazine ring. As
a component of cellular cytoskeleton, vimentin not only provides
support for the integrity of cells but also serves as a platform for the
proper function of various signaling pathways. As a canonical
marker of the epithelial-to-mesenchymal transition, vimentin is
implicated in many diseases and conditions, especially cancer
progression and chemoresistance [23,24]. While vimentin inter-
mediate filament plays important roles in health and disease far
beyond its function as a structural protein [25], it has not been
successfully targeted in drug development. Our study not only
provides unique tools and methods for further research into
vimentin biology, but also open a new venue for targeting vimentin
as a first-in-class therapeutic approach potentially applicable to
various diseases and conditions.
compound
R1
H
R2
IC50
(
m
M)
Vacuole formation
no
R1
H
V21
1.00 0.06
2.20 0.21
3.66 0.55
V22
V23
3-Cl
no
no
3-Cl
3-NO2
3-NO2
package (Fig. 6.). Docking simulations were performed on the
domain of vimentin tetramer (PDB ID: 3KLT) showing two amino
acids, Arg273 and Tyr276, that formed hydrogen bond with nitrogen
atoms on the s-triazine ring of compound V6.
Scheme 1. Synthesis of novel s-triazine analogues V1eV12. Reagents and conditions: (a) MeMgBr, Et2O, 50%; (b) substituted benzaldehydes, conc. HCl, reflux, 67e70%; (c) POCl3,
reflux, 55e59%; (d) K2CO3, Boc2O, THF, water, DMF, rt, 86%; (e) substituted isocyanatobenzenes, CH2Cl2, rt, 52e90%; (f) TFA, CH2Cl2, 49e95%; (g) DIPEA, THF, rt, 79e87%; (h) various
amines, DIPEA, THF, rt, 56e78%.
Scheme 2. Synthesis of amide derivatives V13eV20. Reagents and conditions: (a) 1) LiOHꢁH2O, THF, MeOH; 2) HCl, 59%; (b) Aq. MeNH2, HOBt, EDCI, Et3N, DMF, rt, 59e76%.
5

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Scheme 3. Synthesis of meta-phenylenediamine substituted analogues V21eV23. Reagents and conditions: (a) K2CO3, Boc2O, THF, water, DMF, rt, 86%; (b) 1-Chloro-4-
isocyanatobenzene, CH2Cl2, rt, 90%; (c) TFA, CH2Cl2, 95%; (d) DIPEA, THF, rt, 80e86%; (e) Morpholine, DIPEA, THF, rt, 66e82%.
Fig. 2. Induction of methuosis by compound V6. a. Cytoplasmic vacuolization of U87 cells treated by Compound V6 for 24 h at concentrations ranging from 0.01 to 10 mM. b. Upper,
the moving track of newly formed vacuoles. Lower, a newly-divided cell became vacuolated, rounded and disintegrated.c. Upper, lucifer yellow assay. The cytoplasmic vacuoles were
derived from macropinocytosis. Middle, cells stained with the endoplasmic reticulum (ER) marker. No association of the vacuoles with the ER. Lower, Cells stained with the Golgi
marker. No association of the vacuoles with the Golgi.
4. Experimental section
600 MHz) and 13C NMR (126 MHz or 151 MHz) spectra were meas-
ured on a Brucker APX 400 or Bruker-500 spectrometer using TMS
4.1. Synthesis
as internal standard. Chemical shift values (d) are reported in ppm
relative to tetramethylsilane internal standard. 1H NMR spectra are
represented as follows: chemical shift, multiplicity (s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, br ¼ broad, m ¼ multiplet),
Melting points were obtained using an X4 apparatus and are
uncorrected. Yields refer to isolated products. 1H NMR (400 MHz or
6

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Fig. 3. a: Design process of probe P1 based on the SARs. b: The click reporter S1 (biotin-N3) was previously reported.
coupling constant (J values) in Hz and integration. All reactions
were monitored by thin layer chromatography (TLC), carried out on
silica gel 60 F-254 aluminum sheets using UV, light (254 and
366 nm). Reagents and solvents were available commercially and
purified according to conventional methods.
4.1.1.2. Synthesis of compounds 2a-d. (E)-6-Styryl-1,3,5-triazine-2,4-
diol (2b). 2,4-Dichloro-6-methyl-1,3,5-triazine (1.60 g, 9.76 mmol)
and benzaldehyde (1.03 g, 9.76 mmol) was dissolved in concen-
trated hydrochloric acid (30 mL) and the resulting mixture was
heated to 100 ꢂC for 12 h. The reaction mixture was cooled to room
temperature and diluted with water (60 mL). The crude product
was filtered and the filter cake was washed with ethyl acetate
(10 mL) and cyclohexane (10 mL) to afford 1.66 g (79%) of a white
solid.
4.1.1. General procedure of the synthesis of compounds V1eV12
and V21eV23
4.1.1.1. 2,4-Dichloro-6-methyl-1,3,5-triazine (1).
Methylmagnesium bromide (3.0 mol/l in diethyl ether, 3 mL,
9 mmol) was added dropwise to a solution of 2,4,6-trichloro-1,3,5-
triazine (1.38 g, 7.5 mmol) in THF (8 mL) at ꢀ20 ꢂC under an inert
atmosphere. The reaction was stirred for 2 h at room temperature.
The reaction mixture was quenched with saturated aqueous
ammonium chloride solution (20 mL) and extracted with EtOAc
(20 mL). The organic layer was separated and dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by flash chro-
matography with 10:1 cyclohexane-ethyl acetate (v/v) to afford
4.1.1.3. Synthesis of compounds 3a-d. (E)-2,4-Dichloro-6-styryl-1,3,5-
triazine
(3b). (E)-6-Styryl-1,3,5-triazine-2,4-diol
(794 mg,
3.69 mmol) was dissolved in phosphorus oxychloride (10 mL) and
the resulting mixture was heated to 140 ꢂC for 19 h. The reaction
mixture was cooled to room temperature and diluted with water
(60 mL). The aqueous mixture was then extracted with ethyl ace-
tate (60 mL ꢃ 3) three times. The combined organic layer was
separated and dried over anhydrous sodium sulfate, filtered,
concentrated, and purified by flash chromatography with 20:1
dichloromethane-methanol (v/v) to afford 560 mg of a white solid
615 mg of a white solid (50%). 1H NMR (300 MHz, CDCl3)
3H, CH3).
d: 2.71 (s,
7

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Scheme 4. Synthesis of probe P1. Reagents and conditions: HOBt, EDCI, Et3N, DMF, rt, 76%.
(61%). 1H NMR (300 MHz, DMSO‑d6)
7.68e7.64 (m, 4H), 7.47e7.45 (m, 3H), 7.05 (d, J ¼ 15.9 Hz, 1H) ppm.
d
: 8.32 (d, J ¼ 15.9 Hz, 1H),
4.1.1.5. Synthesis of compound 5a-d. tert-Butyl (4-(3-(4-
chlorophenyl)ureido)phenyl)carbamate (5a).
1-Chloro-4-isocyanatobenzene (461 mg, 3.0 mmol) in dichloro-
methane (5 mL) was added dropwise to a solution of tert-butyl (4-
aminophenyl)carbamate (625 mg, 3.0 mmol) in dichloromethane
(10 mL) at room temperature. The reaction was stirred for 5 h. The
precipitated product was collected by filtration, yielding 977 mg of
a white solid (90%).
4.1.1.4. tert-Butyl (4-aminophenyl)carbamate (4). Di-tert-butyl
dicarbonate (2.18 g, 10.0 mmol) in THF (30 mL) was added dropwise
to a solution of benzene-1,4-diamine (3.24 g, 30.0 mmol) and po-
tassium carbonate (1.52 g, 11.0 mmol) in DMF (10 mL) and water
(5 mL) at room temperature. The reaction was stirred for 4 h. The
reaction mixture was dilute with ethyl acetate and dichloro-
methane (200 mL), and washed with water (100 mL ꢃ 3) three
times. The organic layer was separated and dried over anhydrous
sodium sulfate, filtered, concentrated, and purified by flash chro-
matography with 4:1 cyclohexane-ethyl acetate (v/v) to afford
4.1.1.6. Synthesis of compound 6a-d. 1-(4-Aminophenyl)-3-(4-
chlorophenyl)urea (6a). Trifluoroacetic acid (47 mg, 4.16 mmol) in
dichloromethane(5 mL) was added dropwise to a solution of tert-
butyl
(4-(3-(4-chlorophenyl)ureido)phenyl)carbamate(188 mg,
0.52 mmol) in dichloromethane(10 mL) at room temperature. The
reaction was stirred for 3 h. The reaction mixture was neutralized
1.79 g of a white solid (86%). 1H NMR (300 MHz, CDCl3)
d: 7.14 (d,
J ¼ 8.3 Hz, 2H), 6.65 (d, J ¼ 8.3 Hz, 2H), 6.28 (s, 1H), 3.54 (s, 2H), 1.52
with
a saturated aqueous sodium bicarbonate solution. The
(s, 9H) ppm.
aqueous mixture was then extracted with dichloromethane
(50 mL ꢃ 3) three times. The organic layer was separated and dried
using anhydrous sodium sulfate, concentrated to afford 129 mg of
white solid (95%). 1H NMR (300 MHz, DMSO‑d6)
d: 8.63 (s, 1H, NH);
8.16 (s, 1H, NH); 7.45 (d, J ¼ 8.8 Hz, 2H); 7.29 (d, J ¼ 8.8 Hz, 2H); 7.06
(d, J ¼ 8.6 Hz, 2H); 6.50 (d, J ¼ 8.6 Hz, 2H); 4.80 (s, 2H, eNH2).
4.1.1.7. Synthesis of compound 7a-n. (E)-1-(4-((4-Chloro-6-styryl-1,
3,
5-triazin-2-yl)amino)phenyl)-3-(4-chlorophenyl)urea
(7f).
1-(4-Aminophenyl)-3-(4-chlorophenyl)urea (342 mg, 1.15 mmol),
(E)-2,4-dichloro-6-styryl-1,3,5-triazine (290 mg, 1.15 mmol) and
N,N-diisopropylethylamine (149 mg, 1.15 mmol) were dissolved in
THF (50 mL) under inert atmosphere. The reaction was stirred for
5 h. Half of solvent was removed in vacuo, and the resulting residue
was dilute with water (60 mL). The aqueous mixture was then
extracted with ethyl acetate (120 mL ꢃ 3) three times. The organic
layer was separated and dried using anhydrous sodium sulfate,
concentrated, and added to ethanol (2 mL) with ultrasound in 30 s.
The crude compound was filtered and the filter cake was used
without further purification (yield: 477 mg, 87%).
4.1.1.8. (E)-1-(4-Chlorophenyl)-3-(4-((4-morpholino-6-styryl-1,3,5-
triazin-2-yl)
amino)
phenyl)
urea
(V6).
(E)-1-(4-((4-Chloro-6-styryl-1,3,5-triazin-2-yl)amino)phenyl)-3-
(4-chlorophenyl)urea (248 mg, 0.52 mmol), morpholine (181 mg,
2.08 mmol) and N,N-diisopropylethylamine (202 mg,1.56 mmol)
were dissolved in THF (60 mL) under nitrogen atmosphere. The
reaction was stirred for 4 h. Half of solvents removed in vacuo, and
the resulting residue was diluted with water (100 mL). The aqueous
mixture was then extracted with ethyl acetate (100 mL ꢃ 3) three
Fig. 4. Morphology of V6 and P1 (31.6 nm, 4 days)-induced cytoplasmic vacuolization
in human glioma U87 cells.
8

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Fig. 5. Identification of V6 target proteins. a. Schematic presentation of the procedures and components for the activity-based protein profiling. The chemical structures, functional
groups and reactions of the probe molecule P1 and the click reporter S1 are illustrated. b. P1-binding proteins detected by Western blotting. The two specific proteins were 55KD
and 220 KD in size, respectively. c. The results from MALDI mass spectrometry peptide mapping and sequencing. The sequence of P1-binding protein matched that of human
vimentin.
times. The organic layer was separated and dried using anhydrous
sodium sulfate, concentrated, and added to ethanol (1 mL) with
ultrasound in 30 s. The crude compound was filtered and the filter
cake was dried to afford 189 mg (69%) of a light yellow powder. 1H
compound was prepared analogously as described for V6, starting
from 3-nitrobenzaldehyde and was isolated as a light yellow
powder (69%). 1H NMR (300 MHz, DMSO‑d6)
d: 9.46 (s, 1H), 9.19 (s,
1H), 8.96 (s, 1H), 8.40 (s, 1H), 8.10 (d, J ¼ 8.0 Hz, 2H), 7.95 (d,
J ¼ 15.9 Hz, 1H), 7.62 (d, J ¼ 8.0 Hz, 2H), 7.53e7.58 (m, 1H), 7.39 (d,
J ¼ 8.6 Hz, 2H), 7.31 (d, J ¼ 8.6 Hz, 2H), 7.21 (d, J ¼ 8.6 Hz, 2H), 6.95
(d, J ¼ 15.9 Hz, 1H), 3.73 (m, 4H), 3.59 (m, 4H) ppm. 13C NMR
NMR (300 MHz, DMSO‑d6)d: 9.57 (s, 1H), 8.88 (s, 1H), 8.59 (s, 1H),
7.93 (d, J ¼ 15.9 Hz, 1H), 7.70 (d, J ¼ 8.7 Hz, 2H), 7.65 (d, J ¼ 8.7 Hz,
2H), 7.49 (d, J ¼ 8.7 Hz, 2H), 7.43e7.38 (m, 5H), 7.32 (d, J ¼ 8.7 Hz,
2H), 6.88 (d, J ¼ 15.9 Hz, 1H), 3.82 (m, 4H), 3.68 (m, 4H) ppm. 13
C
(75 MHz, DMSO‑d6) d: 164.34, 163.88, 158.69, 152.79, 148.33, 137.14,
NMR (75 MHz, DMSO‑d6)
d
: 169.99, 164.43, 163.80, 152.46, 138.83,
136.33, 134.58, 133.76, 133.56, 130.31, 130.13, 123.59, 122.39, 120.77,
119.57, 119.46, 118.36, 115.32, 115.03, 65.92, 43.38. HRMS (ESIþ)
calcd for C28H26ClN8O4 [MþH]þ:573.1760, found 573.1750.
138.63, 135.22, 134.17, 134.11, 129.44, 128.90, 128.55, 127.76, 127.16,
125.13, 120.58, 119.58, 118.75, 65.96, 43.37. HRMS(ESI):m/z [MþH]þ
calcd for C28H27ClN7O2þ:528.1909, found 528.1918.
4.1.1.10. (E)-1-(4-Chlorophenyl)-3-(4-((4-(4-methylpiperazin-1-yl)-
6-(3-nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V2). The ti-
tle compound was prepared analogously as described for V6,
4.1.1.9. (E)-1-(4-Chlorophenyl)-3-(4-((4-morpholino-6-(3-
nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V1). The title
9

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
Fig. 6. Docking results of compound V6 and the target protein. a: 3KLT derived from the tetramer vimentin in the PDB database (3KLT is the system number of the protein in the
database); b: used for docking compound V6; c: LibDock docking method, docking 3KLT with V6 compound, the top ten V6 compound conformation score results were obtained; d:
V6 compound scores the first conformation and the protein vimentin action diagram; e: two amino acids: Arg273, Tyr276, and nitrogen atom on the s-triazine ring in compound V6.
starting from 3-nitrobenzaldehyde and using 4-methylpiperazine
for the amination at triazine C4 and was isolated as a light yellow
(300 MHz, DMSO‑d6) d: 9.49 (s, 1H), 8.79 (s, 1H), 8.59 (s, 1H), 8.49 (s,
1H), 8.21 (dd, J ¼ 8.0 Hz, J ¼ 1.8 Hz, 2H), 8.01 (d, J ¼ 15.9 Hz, 1H),
7.77e7.69 (m, 3H), 7.49 (d, J ¼ 8.9 Hz, 2H), 7.39 (d, J ¼ 8.9 Hz, 2H),
7.32 (d, J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 15.9 Hz, 1H), 3.62e3.57 (m, 4H),
powder (66%). 1H NMR (300 MHz, DMSO‑d6)
d: 9.53 (s, 1H), 8.84 (s,
1H), 8.66 (s, 1H), 8.50 (s, 1H), 8.20 (d, J ¼ 7.6 Hz, 2H), 8.03 (d,
J ¼ 15.9 Hz, 1H), 7.73e7.64 (m, 3H), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.41 (d,
J ¼ 8.5 Hz, 2H), 7.31 (d, J ¼ 8.5 Hz, 2H), 7.05 (d, J ¼ 15.9 Hz, 1H), 3.84
(m, 4H), 2.39 (m, 4H), 2.23 (s, 3H) ppm. 13C NMR (75 MHz,
DMSO‑d6) d: 169.53, 164.18, 163.86, 152.48, 148.31, 138.85, 137.15,
136.20, 134.16, 133.55, 130.28, 128.53, 125.09, 123.58, 122.37, 120.60,
1.96e1.94 (m, 4H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 162.87,
152.47, 148.35, 138.85, 137.19, 134.55, 133.83, 133.61, 130.33, 128.55,
125.09, 123.55, 122.24, 120.21, 119.55, 118.75, 45.99, 24.76. HRMS
(ESIþ) calcd for C28H26ClN8O3 [MþH]þ:557.1811, found 557.1816.
119.56, 118.74, 54.33, 45.72, 42.72. HRMS (ESIþ) calcd for
4.1.1.13. (E)-1-(4-Chlorophenyl)-3-(4-((4-(3-hydroxyazetidin-1-yl)-
6-(3-nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V5). The ti-
tle compound was prepared analogously as described for V6,
starting from 3-nitrobenzaldehyde and using azetidin-3-ol for the
amination at triazine C4 and was isolated as a light yellow powder
C
29H29ClN9O3 [MþH]þ:586.2076, found 586.2082.
4.1.1.11. (E)-1-(4-Chlorophenyl)-3-(4-((4-(dimethylamino)-6-(3-
nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V3). The title
compound was prepared analogously as described for V6, starting
from 3-nitrobenzaldehyde and using dimethylamine for the ami-
nation at triazine C4 and was isolated as a yellow powder (67%). 1H
(63%). 1H NMR (300 MHz, DMSO‑d6)
d: 9.56 (s, 1H), 8.88 (s, 1H), 8.69
(s, 1H), 8. 50 (s, 1H), 8.22 (d, J ¼ 8.0 Hz, 2H), 7.98 (d, J ¼ 15.9 Hz, 1H),
7.68e7.74 (m, 3H), 7.49 (d, J ¼ 8.8 Hz, 2H), 7.39 (d, J ¼ 8.8 Hz, 2H),
7.32 (d, J ¼ 8.8 Hz, 2H), 7.08 (d, J ¼ 15.9 Hz, 1H), 5.78 (d, J ¼ 6.6 Hz,
1H), 4.58e4.61 (m, 1H), 4.32e4.37 (m, 2H), 3.86e3.90 (m, 2H) ppm.
NMR (300 MHz, DMSO‑d6) d: 9.50 (s, 1H), 8.81 (s, 1H), 8.62 (s, 1H),
8.50 (s, 1H), 8.21 (dd, J ¼ 8.0 Hz, J ¼ 1.8 Hz, 2H), 8.03 (d, J ¼ 15.9 Hz,
1H), 7.74e7.69 (m, 3H), 7.49 (d, J ¼ 8.9 Hz, 2H), 7.40 (d, J ¼ 8.9 Hz,
2H), 7.32 (d, J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 15.9 Hz, 1H), 3.23e3.17 (m,
13C NMR (75 MHz, DMSO‑d6)
d: 169.36, 165.26, 163.55, 152.51,
148.31, 138.88, 137.09, 136.14, 134.29, 134.05, 133.65, 130.27, 128.54,
125.06, 123.57, 122.25, 120.42, 119.49, 118.67, 60.93, 59.23. HRMS
(ESIþ) calcd for C27H24ClN8O4 [MþH]þ:559.1604, found 559.1609.
6H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 169.19, 164.90, 163.64,
152.48, 148.35, 138.86, 137.19, 135.97, 134.36, 133.98, 133.59, 130.32,
128.55, 125.09, 123.56, 122.29, 120.41, 119.56, 118.74, 35.86. HRMS
(ESIþ) calcd for C28H24ClN8O3 [MþH]þ:531.1654, found 531.1667.
4.1.1.14. (E)-1-(4-Fluorophenyl)-3-(4-((4-morpholino-6-styryl-1,3,5-
triazin-2-yl)amino)phenyl)urea (V7). The title compound was pre-
pared analogously as described for V6, starting from 1-fluoro-4-
isocyanatobenzene and was isolated as a light yellow powder
4.1.1.12. (E)-1-(4-Chlorophenyl)-3-(4-((4-(3-nitrostyryl)-6-(pyrroli-
din-1-yl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V4). The title com-
pound was prepared analogously as described for V6, starting from
3-nitrobenzaldehyde and using pyrrolidine for the amination at
triazine C4 and was isolated as a light yellow powder (62%). 1H NMR
(76%). 1H NMR (300 MHz, DMSO‑d6)
d: 9.65 (s, 1H), 8.78 (s, 1H), 8.66
(s, 1H), 7.95 (d, J ¼ 15.9 Hz, 1H), 7.71 (d, J ¼ 6.6 Hz, 2H), 7.62 (d,
J ¼ 8.5 Hz, 2H), 7.48e7.38 (m, 7H), 7.11 (t, J ¼ 8.5 Hz, 2H), 6.90 (d,
10

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
J ¼ 15.9 Hz, 1H), 3.82 (m, 4H), 3.69 (m, 4H) ppm. 13C NMR (75 MHz,
(4-((4-morpholino-6-styryl-1,3,5-triazin-2-yl)amino)phenyl)ure-
ido)-benzoate (300 mg, 0.54 mmol), THF (10 mL), MeOH (5 mL),
and H2O (1 mL) was added LiOH. H2O (68.5 mg, 1.63 mmol). The
mixture was then heated under reflux for 12 h and concentrated.
H2O (5 mL) was added, and the mixture was then acidified with
2 mol/L HCl to pH ¼ 4. The solid was added to ethanol (2 mL) with
ultrasound in 30 s, filtered, and dried to give the product as a light
yellow solid, which was taken to next step without purification
DMSO‑d6) d: 172.45, 170.73, 165.41, 158.07, 155.72, 152.65, 146.33,
140.59, 136.86, 135.98, 134.83, 129.84, 128.89,128.09, 125.96, 121.90,
119.89, 118.93, 115.37, 115.08, 65.66, 59.23. HRMS (ESIþ) calcd for
C
28H27FN7O2 [MþH]þ:512.2205, found 512.2215.4.1.1.15. (E)-1-(4-
((4-(3-Chlorostyryl)-6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)-
3-(4-fluorophenyl)urea (V8). The title compound was prepared
analogously as described for V6, starting from 1-fluoro-4-
isocyanatobenzene and was isolated as a light yellow powder
(171 mg, 59% yield). 1H NMR (300 MHz, DMSO‑d6)
d: 12.33 (s, 1H),
(66%). 1H NMR (300 MHz, DMSO‑d6)
d: 9.56 (s, 1H), 8.83 (s, 1H), 8.70
9.59 (s, 1H), 9.41 (s, 1H), 9.09 (s, 1H), 7.94 (d, J ¼ 15.9 Hz, 1H), 7.88 (d,
J ¼ 8.3 Hz, 2H), 7.77e7.63 (m, 4H), 7.59 (d, J ¼ 8.3 Hz, 2H), 7.51e7.36
(m, 5H), 6.89 (d, J ¼ 15.9 Hz, 1H), 3.83 (s, 4H), 3.69 (s, 4H) ppm.
(s, 1H), 7.91 (d, J ¼ 15.9 Hz, 1H), 7.81 (s, 1H), 7.63e7.69 (m, 3H),
7.44e7.49 (m, 4H), 7.40 (d, J ¼ 8.8 Hz, 2H), 7.12 (t, J ¼ 8.8 Hz, 2H),
6.95 (d, J ¼ 15.9 Hz, 1H), 3.82 (m, 4H), 3.69 (m, 4H) ppm. 13C NMR
(75 MHz, DMSO‑d6)
d
: 169.74, 164.37, 163.83, 158.75, 155.60, 152.72,
4.1.2.2. (E)-N-(2-(Dimethylamino)ethyl)-4-(3-(4-((4-morpholino-6-
137.54, 137.07, 136.23, 134.43, 133.91, 133.72, 130.63, 129.00, 127.36,
126.32, 120.67, 119.72, 119.62, 118.55, 115.33, 115.03, 65.95, 43.35.
HRMS (ESIþ) calcd for C28H26ClFN7O2 [MþH]þ:546.1815, found
546.1821.
styryl-1,3,5-triazin-2-yl)amino)phenyl)ureido)benzamide
(V16).
A solution of (E)-4-(3-(4-((4-morpholino-6-styryl-1,3,5-triazin-2-
yl) amino) phenyl) ureido)benzoic acid (8, 100 mg, 0.186 mmol),
HOBt (32.7 mg, 0.242 mmol), EDCI (53.5 mg, 0.279 mmol), N1,N1-
dimethylethane-1,2-diamine (21.3 mg, 0.242 mmol) and triethyl-
amine (37.6 mg, 0.372 mmol) in 2 mL of DMF was stirred for 8 h at
room temperature. At the end, CH2Cl2 (40 mL) was added and
washing was done with saturated NaHCO3 and H2O. The mixture
was concentrated and purified by silica gel chromatography, with
CH2Cl2/methanol (20:1, v/v) to give the product as a light yellow
4.1.1.15. (E)-1-(4-((4-(4-Chlorostyryl)-6-morpholino-1,3,5-triazin-2-
yl)amino)phenyl)-3-(4-fluorophenyl)urea (V9). The title compound
was prepared analogously as described for V6, starting from 1-
fluoro-4-isocyanatobenzene and 4-chlorobenzaldehyde and was
isolated as a light yellow powder (75%). 1H NMR (300 MHz,
DMSO‑d6)
d
: 9.6 (s, 1H), 8.66 (s, 1H), 8.54 (s, 1H), 7.88 (d, J ¼ 15.9 Hz,
solid (86 mg, 76% yield). 1H NMR (300 MHz, DMSO‑d6)
d: 9.64 (s,
1H), 7.75 (d, J ¼ 8.4 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.43e7.49 (m,
4H), 7.38 (d, J ¼ 8.8 Hz, 2H), 7.11 (t, J ¼ 8.8 Hz, 1H), 6.89 (d,
J ¼ 15.9 Hz, 1H), 3.82 (m, 4H), 3.68 (m, 4H) ppm. 13C NMR (75 MHz,
1H), 9.11 (s, 1H), 8.84 (s, 1H), 8.37 (t, J ¼ 5.4 Hz, 1H), 7.99 (d,
J ¼ 15.9 Hz, 1H), 7.85 (d, J ¼ 8.5 Hz, 2H), 7.77e7.70 (m, 4H), 7.58 (d,
J ¼ 8.5 Hz, 2H), 7.52e7.45 (m, 5H), 6.93 (d, J ¼ 15.9 Hz, 1H), 3.88 (m,
4H), 3.74 (m, 4H), 3.49e3.45 (m, 2H), 2.67 (t, J ¼ 6.1 Hz, 2H), 2.41 (s,
DMSO‑d6) d: 169.76, 164.37, 163.83, 158.77, 155.60, 152.72, 137.54,
137.09, 136.23, 134.43, 133.95, 133.72, 130.63, 129.05, 127.36, 126.36,
120.67, 119.72, 119.62, 118.58, 115.33, 115.03, 65.88, 43.37. HRMS
(ESIþ) calcd for C28H26ClFN7O2 [MþH]þ:546.1815, found 546.1822.
6H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 165.83, 164.45, 163.81,
152.38, 142.63, 138.65, 134.21, 134.08, 129.44, 128.91, 128.13, 127.75,
127.19, 120.59, 118.70, 116.90, 65.96, 57.73, 44.50, 43.37, 36.55.
HRMS (ESIþ) calcd for
C
33H38N9O3 [MþH]þ:608.3092, found
4.1.1.16. (E)-1-(4-((4-(4-Chlorostyryl)-6-morpholino-1,3,5-triazin-2-
yl)amino)phenyl)-3-(3-fluorophenyl)urea (V10). The title com-
pound was prepared analogously as described for V6, starting from
1-fluoro-3-isocyanatobenzene and 4-chlorobenzaldehyde and was
isolated as a light yellow powder (77%). 1H NMR (300 MHz,
608.3104.
4.1.2.3. (E)-N,N-Dimethyl-4-(3-(4-((4-morpholino-6-styryl-1,3,5-
triazin-2-yl)amino)phenyl)ureido)benzamide (V13). The title com-
pound was prepared analogously as described for V16, using
dimethylamine(40 wt% in H2O) for the amine/acid coupling reac-
tion and was isolated as a light yellow powder (76%). 1H NMR
DMSO‑d6)
d: 9.60 (s, 1H), 8.86 (s, 1H), 8.62 (s, 1H), 7.91 (d,
J ¼ 15.9 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz, 2H), 7.53e7.58 (m, 1H), 7.39 (d,
J ¼ 8.6 Hz, 2H), 7.31 (d, J ¼ 8. 6 Hz, 2H), 7.21 (d, J ¼ 8.6 Hz, 2H), 6.95
(d, J ¼ 15.9 Hz, 1H), 3.73 (m, 4H), 3.59 (m, 4H) ppm. 13C NMR
(300 MHz, DMSO‑d6) d: 9.60 (s, 1H), 8.86 (s, 1H), 8.64 (s, 1H), 7.94 (d,
J ¼ 15.9 Hz, 1H), 7.72e7.65 (m, 4H), 7.51 (d, J ¼ 8.5 Hz, 2H),
7.46e7.40 (m, 5H), 7.36 (d, J ¼ 8.5 Hz, 2H), 6.89 (d, J ¼ 15.9 Hz, 1H),
3.83 (m, 4H), 3.69 (m, 4H), 2.97 (s, 6H) ppm. 13C NMR (75 MHz,
(75 MHz, DMSO‑d6) d: 169.8, 164.0, 152.4, 141.8, 141.6, 137.3, 134.2,
134.0, 133.9, 130.3, 130.2, 129.5, 128.9, 120.6, 118.8, 113.7, 108.0,
107.8, 104.8, 104.5, 65.9, 43.3. HRMS (ESIþ) calcd for C28H26ClFN7O2
[MþH]þ:546.1815, found 546.1823.
DMSO‑d6) d: 170.0, 164.4, 163.8, 152.4, 141.0, 138.7, 135.2, 134.1,
129.5, 129.2, 128.9, 128.2, 127.8, 120.6, 118.7, 117.2, 66.0, 43.4. HRMS
(ESIþ) calcd for C31H33N8O3 [MþH]þ:565.1670, found 565.1676.
4.1.1.17. (E)-1-(4-Fluorophenyl)-3-(4-((4-morpholino-6-(3-
nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V11). The title
compound was prepared analogously as described for V6, starting
from 1-fluoro-4-isocyanatobenzene and 3-nitrobenzaldehyde and
was isolated as a light yellow powder (66%). 1H NMR (300 MHz,
4.1.2.4. (E)-1-(4-(4-Methylpiperidine-1-carbonyl)phenyl)-3-(4-((4-
morpholino-6-styryl-1,3,5-triazin-2-yl)amino)phenyl)urea
The title compound was prepared analogously as described for V16,
using 4-methylpiperidine for the amine/acid coupling reaction and
was isolated as a light yellow powder (65%). 1H NMR (300 MHz,
(V14).
DMSO‑d6) d: 9.58 (s, 1H), 8.84 (s, 1H), 8.72 (s, 1H), 8.52 (s, 1H), 8.22
(d, J ¼ 8.0 Hz, 2H), 8.06 (d, J ¼ 15.9 Hz, 1H), 7.74 (d, J ¼ 8.0 Hz, 1H),
7.63e7.69 (m, 2H), 7.44e7.49 (m, 2H),7.40 (d, J ¼ 8.8 Hz, 2H), 7.13 (d,
J ¼ 8.8 Hz, 2H), 7.07 (d, J ¼ 15.9 Hz, 1H), 3.84 (m, 4H), 3.70 (m, 4H)
DMSO‑d6) d: 9.56 (s, 1H), 8.83 (s, 1H), 8.62 (s, 1H), 7.94 (d,
J ¼ 15.9 Hz,1H), 7.71e7.64 (m, 4H), 7.50 (d, J ¼ 8.5 Hz, 2H), 7.44e7.39
(m, 5H), 7.30 (d, J ¼ 8.5 Hz, 2H), 6.88 (d, J ¼ 15.9 Hz, 1H), 3.82 (m,
4H), 3.69 (m, 4H), 3.33 (m, 2H), 2.89 (m, 1H), 2.73 (m, 1H), 1.63e1.61
(m, 3H), 1.12e1.01 (m, 2H), 0.92 (d, J ¼ 6.1 Hz, 3H) ppm. 13C NMR
ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 164.33, 163.86, 158.69, 152.79,
148.33, 137.14, 136.31, 134.58, 133.76, 133.56, 130.31, 130.13, 123.60,
122.39, 120.71, 119.57, 119.46, 118.39, 115.32, 115.03, 65.96, 43.36.
HRMS (ESIþ) calcd for C28H26FN8O4 [MþH]þ:557.2056, found
557.2060.
(75 MHz, DMSO‑d6) d: 168.85, 164.44, 163.81, 152.42, 140.92, 135.22,
134.18, 134.11, 129.25, 128.91, 128.08, 127.87, 127.76, 120.59, 118.72,
117.34, 65.96, 43.37, 30.47, 21.57. HRMS (ESIþ) calcd for C35H39N8O3
[MþH]þ:619.3140, found 619.3151.
4.1.2. General procedure of the synthesis of compounds V13eV20
4.1.2.1. (E)-4-(3-(4-((4-Morpholino-6-styryl-1,3,5-triazin-2-yl)
amino)phenyl)ureido)benzoic acid (8). To a stirred mixture of 4-(3-
4.1.2.5. (E)-1-(4-(4-Methylpiperazine-1-carbonyl)phenyl)-3-(4-((4-
morpholino-6-styryl-1,3,5-triazin-2-yl)amino)phenyl)urea
(V15).
11

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
The title compound was prepared analogously as described for V16,
using 1-methylpiperazine for the amine/acid coupling reaction and
was isolated as a light yellow powder (66%). 1H NMR (300 MHz,
J ¼ 5.2 Hz, 1H), 7.94 (d, J ¼ 15.9 Hz, 1H), 7.78 (d, J ¼ 8.5 Hz, 2H),
7.65e7.72 (m, 4H), 7.52 (d, J ¼ 8.5 Hz, 2H), 7.40e7.46 (m, 5H), 7.34
(d, J ¼ 8.5 Hz, 2H), 6.89 (d, J ¼ 15.9 Hz, 1H), 3.83 (m, 4H), 3.69 (m,
DMSO‑d6)
d
: 9.60 (s, 1H), 8.91 (s, 1H), 8.67 (s, 1H), 7.95 (d,
4H), 3.56e3.59 (m, 4H), 3.35 (m, 2H), 2.43e2.50 (m, 6H) ppm. 13
C
J ¼ 15.9 Hz,1H), 7.65e7.72 (m, 4H), 7.52 (d, J ¼ 8.5 Hz, 2H), 7.41e7.43
NMR (75 MHz, DMSO‑d6) d: 169.99, 165.67, 164.41, 163.78, 152.34,
(m, 5H), 7.34 (d, J ¼ 8.5 Hz, 2H), 6.89 (d, J ¼ 15.9 Hz, 1H), 3.83 (m,
142.49, 138.67, 135.19, 134.23, 134.01, 129.45, 128.91, 128.08, 127.76,
127.40, 127.12, 120.56, 118.77, 116.98, 66.15, 65.96, 57.44, 53.27,
43.35, 36.41. HRMS (ESIþ) calcd for C35H40N9O4 [MþH]þ:650.3198,
found 650.3212.
4H), 3.69 (m, 4H), 3.50 (m, 4H), 2.33 (m, 4H), 2.21 (s, 3H)ppm. 13
C
NMR (75 MHz, DMSO‑d6) d: 169.98, 168.92, 164.41, 152.41, 141.17,
138.65, 135.20, 134.18, 134.09, 129.45, 128.91, 128.55, 128.18, 127.76,
127.14, 120.57, 118.71, 117.30, 65.96, 54.78, 53.58, 47.60, 45.50, 43.38.
HRMS (ESIþ) calcd for
C
34H38N9O3 [MþH]þ:620.3092, found
4.1.3. General procedure of the synthesis of compounds V21eV23
4.1.3.1. (E)-1-(4-Chlorophenyl)-3-(3-((4-morpholino-6-styryl-1,3,5-
triazin-2-yl)amino)phenyl)urea (V21). The title compound was
prepared analogously as described for V6, starting from benzene-
1,3-diamine and was isolated as a light yellow powder (78%). 1H
620.3105.
4.1.2.6. (E)-N-(3-(Dimethylamino)propyl)-4-(3-(4-((4-morpholino-
6-styryl-1,3,5-triazin-2-yl)amino)phenyl)ureido)benzamide (V17).
The title compound was prepared analogously as described for V16,
using N1, N1-dimethylpropane-1, 3-diamine for the amine/acid
coupling reaction and was isolated as a light yellow powder (61%).
NMR (300 MHz, DMSO‑d6) d: 9.63 (s, 1H), 8.81 (s, 1H), 8. 70 (s, 1H),
8. 32 (s, 1H) 8.01 (d, J ¼ 15.9 Hz, 1H), 7.69e7.71 (m, 2H), 7.49 (d,
J ¼ 8.8 Hz, 2H), 7.39 (m, 3H), 7.33 (d, J ¼ 8.8 Hz, 2H), 7.25e7.16 (m,
2H), 6.93 (m,1H), 6.89 (d, J ¼ 15.9 Hz,1H), 3.87 (m, 4H), 3.69 (m, 4H)
1H NMR (300 MHz, DMSO‑d6)
d: 9.61 (s, 1H), 9.02 (s, 1H), 8.77 (s,
1H), 8.37 (t, J ¼ 5.4 Hz, 1H), 7.94 (d, J ¼ 15.9 Hz, 1H), 7.77 (d,
J ¼ 8.5 Hz, 2H), 7.72e7.64 (m, 4H), 7.52 (d, J ¼ 8.5 Hz, 2H), 7.43e7.41
(m, 5H), 6.88 (d, J ¼ 15.9 Hz, 1H), 3.82 (m, 4H), 3.69 (m, 4H),
3.29e3.24 (m, 2H), 2.27 (t, J ¼ 6.9 Hz, 2H), 2.15 (s, 6H), 1.69e1.62 (m,
ppm. 13C NMR(75 MHz, DMSO‑d6)
d: 170.09, 164.44, 163.90, 152.30,
140.28, 139.66, 138.92, 138.71, 135.25, 129.42, 128.85, 128.55, 127.77,
127.06, 125.28, 119.68, 113.68, 112.19, 109.91, 66.03, 43.41. HRMS
(ESIþ) calcd for C28H27ClN7O2 [MþH]þ:528.1909, found 528.1916.
2H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 165.6, 164.4, 152.4, 142.5,
135.2,134.2,134.1,129.5,128.9,128.0,127.8,127.5,120.6,118.7,116.9,
65.9, 56.9, 45.1, 43.3, 37.6, 27.1. HRMS (ESIþ) calcd for C34H40N9O8
[MþH]þ:662.3249, found 662.3255.
4.1.3.2. (E)-1-(4-Chlorophenyl)-3-(3-((4-(3-chlorostyryl)-6-
morpholino-1,3,5-triazin-2-yl)amino)phenyl)urea (V22). The title
compound was prepared analogously as described for V6, starting
from benzene-1,3-diamine and 3-chlorobenzaldehyde and was
isolated as a light yellow powder (69%). 1H NMR (300 MHz,
4.1.2.7. (E)-4-(3-(4-((4-Morpholino-6-styryl-1,3,5-triazin-2-yl)
amino)phenyl)ureido)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide (V18).
The title compound was prepared analogously as described for V16,
using 2-(pyrrolidin-1-yl)ethanamine for the amine/acid coupling
reaction and was isolated as a light yellow powder (66%). 1H NMR
DMSO‑d6) d: 9.64 (s, 1H), 8.82 (s, 1H), 8.71 (s, 1H), 8.31 (s, 1H) 7.98
(d, J ¼ 15.9 Hz, 1H), 7.80 (s,1H), 7.68 (d, J ¼ 7.0 Hz,1H), 7.43e7.50 (m,
4H), 7.32 (d, J ¼ 8.8 Hz, 2H), 7.16e7.26 (m, 2H), 6.96 (d, J ¼ 15.9 Hz,
1H), 6.94 (d, J ¼ 7.0 Hz, 1H), 3.88 (m, 4H), 3.69 (m, 4H) ppm. 13C
(300 MHz, DMSO‑d6)
d: 9.61 (s, 1H), 9.51 (s, 1H), 9.23 (s, 1H), 8.43 (s,
NMR (75 MHz, DMSO‑d6) d: 169.84, 164.38, 163.91, 152.30, 140.20,
1H), 7.95 (d, J ¼ 15.9 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 2H), 7.69 (dd,
J ¼ 17.5, 7.5 Hz, 4H), 7.54 (d, J ¼ 8.5 Hz, 2H), 7.43 (d, J ¼ 4.6 Hz, 5H),
6.89 (d, J ¼ 15.7 Hz, 1H), 3.83 (s, 4H), 3.70 (s, 4H), 3.44 (d, J ¼ 5.4 Hz,
2H), 2.83 (d, J ¼ 4.5 Hz, 6H), 1.76 (s, 4H) ppm. 13C NMR (75 MHz,
139.67, 138.72, 137.55, 137.36, 133.71, 130.57, 129.01, 128.76, 128.61,
128.53, 127.32, 126.37, 125.25, 119.62, 113.72, 112.23, 109.94, 66.02,
43.40. HRMS (ESIþ) calcd for C28H26Cl2N7O2 [MþH]þ:562.1520,
found 562.1530.
DMSO‑d6) d: 169.95, 165.83, 164.41, 163.77, 152.51, 142.80, 138.66,
135.21, 134.26, 134.05, 129.44, 128.91, 128.19, 127.76, 127.15, 126.95,
124.86, 120.59, 118.41, 116.69, 65.96, 54.46, 53.48, 45.44, 43.35,
37.46, 29.27, 29.00, 22.89. HRMS (ESIþ) calcd for C35H40N9O3
[MþH]þ:634.3249, found 634.3264.
4.1.3.3. (E)-1-(4-Chlorophenyl)-3-(3-((4-morpholino-6-(3-
nitrostyryl)-1,3,5-triazin-2-yl)amino)phenyl)urea (V23). The title
compound was prepared analogously as described for V6, starting
from benzene-1,3-diamine and 3-nitrobenzaldehyde and was iso-
lated as a light yellow powder (66%). 1H NMR (300 MHz, DMSO‑d6)
d:
4.1.2.8. (E)-4-(3-(4-((4-Morpholino-6-styryl-1,3,5-triazin-2-yl)
amino)phenyl)ureido)-N-(2-(piperidin-1-yl)ethyl)benzamide (V19).
The title compound was prepared analogously as described for V16,
using 2-(piperidin-1-yl)ethanamine for the amine/acid coupling
reaction and was isolated as a light yellow powder (67%). 1H NMR
9.62 (s, 1H), 8.83 (s, 1H), 8.72 (s, 1H), 8.47 (s, 1H) 8.30 (s, 1H) 8.18 (d,
J ¼ 8.0 Hz, 2H), 8.11 (d, J ¼ 15.9 Hz, 1H), 7.65 (t, J ¼ 7.0 Hz, 1H) 7.45 (d,
J ¼ 8.0 Hz, 2H), 7.14e7.32 (m, 4H), 7.05 (d, J ¼ 15.9 Hz, 1H), 6.93 (t,
J ¼ 7.0 Hz, 1H), 3.87 (m, 4H), 3.67 (m, 4H) ppm. 13C NMR (75 MHz,
DMSO‑d6) d: 169.63, 164.37, 163.94, 152.32, 148.28, 140.18, 139.95,
(300 MHz, DMSO‑d6)
d
: 9.66 (s, 1H), 9.50 (s, 1H), 9.22 (s, 1H), 8.52 (s,
139.70, 138.72, 137.13, 136.59, 133.59, 130.21, 129.96, 128.60, 128.49,
125.24, 123.56, 122.31, 119.59, 113.72, 112.22, 109.91, 66.03, 43.42,
30.37. HRMS (ESIþ) calcd for C28H26Cl2N8O4 [MþH]þ:573.1687, found
573.1760.
1H), 7.99 (d, J ¼ 15.9 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 2H), 7.77e7.70 (m,
4H), 7.60 (d, J ¼ 8.5 Hz, 2H), 7.49e7.43 (m, 5H), 6.94 (d, J ¼ 15.9 Hz,
1H), 3.87 (m, 4H), 3.74 (m, 4H), 3.54e3.52 (m, 2H), 2.80 (m, 6H),
1.66 (m, 4H), 1.49 (m, 2H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d:
169.98, 165.91, 164.40, 163.76, 152.47, 142.80, 138.62, 135.19, 134.19,
134.10, 129.43, 128.91, 128.20, 127.75, 127.12,126.92, 120.58, 118.49,
116.76, 65.96, 56.77, 53.26, 43.35, 35.56,24.15, 22.77. HRMS (ESIþ)
calcd for C36H42N9O3 [MþH]þ:648.3405, found 648.3423.
4.1.4. Synthetic procedures of probe P1 (E)-N-(2-(3-(But-3-yn-1-
yl)-3H-diazirin-3-yl)ethyl)-4-(3-(4-((4-morpholino-6-styryl-1,3,5-
triazin-2-yl)amino)phenyl)ureido)benzamide (P1)
A solution of (E)-4-(3-(4-((4-morpholino-6-styryl-1,3,5-triazin-
2-yl)amino)phenyl)ureido) benzoic acid (8, 100 mg, 0.186 mmol),
HOBt (32.7 mg, 0.242 mmol), EDCI (53.5 mg, 0.279 mmol), 2-(3-
4.1.2.9. (E)-4-(3-(4-((4-Morpholino-6-styryl-1,3,5-triazin-2-yl)
amino)phenyl)ureido)-N-(2-morpholinoethyl)benzamide
(V20).
(but-3-yn-1-yl)-3H-diazirin-3-yl)ethanamine
(L2,
33.0 mg,
The title compound was prepared analogously as described for V16,
using 2-morpholinoethanamine for the amine/acid coupling reac-
tion and was isolated as a light yellow powder (68%). 1H NMR
0.242 mmol) and triethylamine (37.6 mg, 0.372 mmol) in 2 mL of
DMF was stirred for 8 h at room temperature. At the end, CH2Cl2
(40 mL) was added and washing was done with saturated NaHCO3
and H2O. The mixture was concentrated and purified by silica gel
(300 MHz, DMSO‑d6) d: 9.60 (s, 1H), 8.93 (s, 1H), 8.68 (s, 1H), 8.27 (t,
12

L. Zhang, Z. Qu, J. Wu et al.
European Journal of Medicinal Chemistry 214 (2021) 113188
chromatography, with CH2Cl2/methanol (15:1, v/v) to give the
product as a light yellow solid (92.8 mg, 76% yield). 1H NMR
by MALDI Mass Spectrometry peptide mapping and sequencing
(MALDI-MS/MS performed by Alphalyse, Inc.).
(300 MHz, DMSO‑d6) d: 9.62 (s, 1H), 8.94 (s, 1H), 8.68 (s, 1H), 8.34 (t,
J ¼ 5.2 Hz, 1H), 7.96 (d, J ¼ 15.9 Hz, 1H), 7.80 (d, J ¼ 8.6 Hz, 2H),
7.67e7.74 (m, 4H), 7.55 (d, J ¼ 8.6 Hz, 2H), 7.42e7.48 (m, 5H), 6.90
(d, J ¼ 15.9 Hz, 1H), 3.84 (m, 4H), 3.71 (m, 4H), 3.13e3.20 (m, 2H),
2.87 (d, J ¼ 2.5 Hz, 1H), 2.04 (td, J ¼ 7.4 Hz, J ¼ 2.5 Hz, 2H), 1.62e1.69
4.4. Molecular docking
The crystal structure of vimentin fragment 3KLT from the Pro-
tein Data Bank and LibDock module in Discovery Studio were
employed for the docking study. Based on the ligand of 3KLT, the
active pocket was set with a sphere radius of 10 Å. The number of
grid points was set to 1000; the conformation method, to BEST;
maximum number of conformations, to 255; and all other param-
eters, to the default values. The resulting poses with 10 highest
LibDock scores were investigated.
(m, 4H) ppm. 13C NMR (75 MHz, DMSO‑d6)
d: 170.05, 165.66, 164.41,
163.87, 152.16, 142.45, 140.30, 139.59, 138.91, 135.22, 129.44, 128.87,
128.63, 128.07, 127.77, 127.40, 127.01, 117.02, 113.69, 112.13, 109.78,
83.13, 71.72, 66.05, 43.41, 34.27, 31.99, 31.30, 27.28, 12.67. ES-MS m/
z: 655.5[M-H]-, C36H36N10O3(MW ¼ 656.7).
4.2. Cell culture, cell proliferation assay and phenotypic screening
Author information
Human cancer cell lines were obtained from the cell bank of
Chinese Academy of Sciences or from ATCC. The cells were cultured
L.Z. and Q.Z. are partially supported by Nanjing Shiqi Pharma-
ceutical Co. Ltd.. S.Y. is a co-founder of Shiqi Pharmaceutical Co. Ltd..
Z.Q. and J.W. are partially supported by Luoda Biosciences, Inc., L.M.
is a co-founder of Aluda Pharmaceuticals, Inc. R.C. holds an adjunct
position at Soochow University, is an employee of Aluda Pharma-
ceuticals, Inc. and the appointed legal representative of Luoda
Biosciences, Inc. T.Z., Q.Y., and Y.X. declare no competing financial
interests. Readers are welcome to comment on the online version of
the paper. Correspondence and requests for materials should be
addressed to Q.Y (qz_yao@163.com), or Y.X. (yingxu@suda.edu.cn),
or R.C. (ruihuan@aludapharm.com).
in RMPI1640 supplemented with 10% fetal bovine serum, 2 mM
glutamine and 1x Penicillin-Streptomycin (100 IU/mL-100
L-
g/mL)
m
at 37 ꢂC in 5% CO2 incubator. For proliferation assay, the cells were
seeded in the wells of 96-well plates at densities of
1500e3000 cells/well, depending on the line of cells, in the pres-
ence of control vehicles or testing compounds at final concentra-
tions of 0, 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16, 10, 31.6 mM and cultured
for 4 days. The cell viability was measured by CellTiter-Glo® assay
(Promega). IC50s were calculated as mean SD from results of
triplicate experiments. The cytoplasmic vacuolization was first
visually screened under microscope on U87 cells treated with in-
dividual testing compounds at 1
individual positive compounds were then titrated down to the
lowest concentration of 0.01 M. The methuosis-inducing abilities
of individual compounds were scored based on their minimal
effective concentration in U87 cells.
mM. The minimal effective doses of
Declaration of competing interest
m
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.3. Activity-based protein profiling
Acknowledgements
U87 cells at exponential growth phase were treated with 0.1 mìM
We are grateful to H.S. Phillips, J.P. Henschke, JY Wang and PY
Wang for helpful discussions and advice with manuscript prepa-
ration. This work was supported by grants from the National Sci-
ence Foundation of China (31230049 Y.X), Royal Society-Newton
Advanced Fellowship (NA150373 to Y.X), and a project funded by
the Priority Academic Development of Jiangsu Higher Education
Institutions.
of the probe molecule P1 or with DMSO of the same volume for 3 h,
then washed twice with cold-PBS. The flask of the cells was added in
a minimal volume of cold PBS (10ml/150 cm2), and placed on the top
of irradiator from which the cells got UV irradiation (315e385 nm)
through the bottom of the flasks for 30 min. The cells were trypsi-
nized and pelleted by centrifugation, washed twice with cold PBS, re-
suspended in Hepes lysis buffer (25 mM Hepes, 150 mM NaCl, 2 mM
MgCl2, 0.1% NP-40, Protease inhibitors, pH 7.5), homogenized by a
brief sonication and rotated at 4 ꢂC for 30 min, followed by centri-
fugation at top speed at 4 ꢂC for 10 min. The cell lysates were pre-
cleared with Agarose-streptavidin by incubation at 4 ꢂC for 2 h or
overnight. Added to the pre-cleared cell lysates with premixed-
cocktail freshly prepared from stock solutions to a final concentra-
tion of 1 mM CuSO4, 1 mM TCEP [tris(2-carboxyethyl)phosphine],
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113188.
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