Design, synthesis, docking, molecular dynamics and bioevaluation studies on novel N-methylpicolinamide and thienopyrimidine derivatives with inhibiting NF-κB and TAK1 activities: Cheminformatics tools RDKit applied in drug design

Article abstract of DOI:10.1016/j.ejmech.2021.113576

Using cheminformatics tools RDKit and literature investigation, four series of 24 thienopyrimidine/N-methylpicolinamide derivatives substituted with pyrimidine were designed, synthesized and evaluated for activities against three cancer cell lines (MDA-MB-231, HCT116 and A549), TAK1 kinase and NF-κB signaling pathway. Almost all compounds showed selectivity toward the A549 cell lines and the most promising compound 38 could inhibit TAK1 kinase and NF-κB signaling pathway with the IC₅₀ values of 0.58 and 0.84 μM. Moreover, 38 can induce cell cycle arrest of A549 cells at the G2/M checkpoint with 30.57% and induce apoptosis (34.94%) in a concentration-dependent manner. And western blot showed that compound 38 could inhibit TNF-α-induced IκBα phosphorylation, IκBα degradation, p65 phosphorylation and TAK1 phosphorylation, and reduce the expression of p65. What's more, the studies of docking, molecular dynamics, MM/PBSA and frequency analysis theoretically supported the conclusions of the bioevaluation.

Full text of DOI:10.1016/j.ejmech.2021.113576

European Journal of Medicinal Chemistry 223 (2021) 113576
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, docking, molecular dynamics and bioevaluation
studies on novel N-methylpicolinamide and thienopyrimidine
derivatives with inhibiting NF-kB and TAK1 activities:
Cheminformatics tools RDKit applied in drug design
Linxiao Wang 1, Qian Zhang b 1, Zhe Wang , Wufu Zhu
a
,
,
a
b
,
**
, Ninghua Tan a
, *
a
State Key Laboratory of Natural Medicines, Department of TCMs Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing, 211198, China
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science &Technology Normal University, Nanchang, 330013,
b
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 February 2021
Received in revised form
Using cheminformatics tools RDKit and literature investigation, four series of 24 thienopyrimidine/N-
methylpicolinamide derivatives substituted with pyrimidine were designed, synthesized and evaluated
for activities against three cancer cell lines (MDA-MB-231, HCT116 and A549), TAK1 kinase and NF-
signaling pathway. Almost all compounds showed selectivity toward the A549 cell lines and the most
promising compound 38 could inhibit TAK1 kinase and NF- B signaling pathway with the IC50 values of
M. Moreover, 38 can induce cell cycle arrest of A549 cells at the G2/M checkpoint with
0.57% and induce apoptosis (34.94%) in a concentration-dependent manner. And western blot showed
-induced I phosphorylation, I degradation, p65 phos-
kB
18 May 2021
Accepted 19 May 2021
Available online 5 June 2021
k
0
3
.58 and 0.84 m
Keywords:
that compound 38 could inhibit TNF-
a
k
Ba
kBa
Cheminformatics
N-methylpicolinamide
Thienopyrimidine
phorylation and TAK1 phosphorylation, and reduce the expression of p65. What's more, the studies of
docking, molecular dynamics, MM/PBSA and frequency analysis theoretically supported the conclusions
of the bioevaluation.
NF-kB
TAK1
Molecular dynamics
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
an association between dysregulated expression of TAK1 and many
human diseases, including cancer [9]. Therefore, NF-kB signaling
Nuclear factor
factors, was discovered as a protein in B cells [1]. NF-
k
B (NF-
k
B), a family of important transcription
B signaling
pathway and TAK1 kinase are becoming an attractive therapeutic
target for cancer treatment.
k
pathway, showing a critical role in many biological processes,
controls the expression of over 500 target genes, which involved
into cell proliferation, apoptosis, angiogenesis, metastasis, and
inflammation [2,3]. Abnormal activation of NF-kB pathway is
frequently happened in many diseases, such as cancer, arthritis, and
NF-
kB inhibitors include synthetic compounds, such as potential
NF- B inhibitors QNZ (EVP4593) [10], SC75741 [11] and CBL-0137
k
[12], and natural inhibitors such as Caffeic Acid Phenethyl Ester
[13], RA-VII, RA-V and RA-XII [14e16]. Although many drugs come
from organic synthesis, natural products, an important source of
lead compounds, have played a vital role in drug discovery for
centuries [17,18]. Moreover, these compounds displayed in Fig. 1,
showed three obvious structural characteristics: HD/HR (hydrogen
bond donor and hydrogen bond receptor), HI (hydrophobic inter-
action/hydrophilic interaction) and a 2e3 atom long linker. Due to
diabetes [4e7]. Transforming growth factor beta-activated kinase 1
(
TAK1/MAP3K7), an evolutionarily conserved member of the
mitogen-activated protein kinase family, is a key kinase upstream
of NF- B, which can be activated by various stimuli, and plays an
essential role in this pathway [8]. Increasing studies have suggested
k
the NF-kB is associated with various cancers [4], it was taken into
consideration to using cheminformatics tools RDKit to analysis the
top skeletons in the anticancer compounds library (contained 2547
anticancer compounds, which were obtained from the library of the
web https://www.selleck.cn) and the results were summarized in
*
Corresponding author.
** Corresponding author.
E-mail addresses: zhuwufu.1122@163.com (W. Zhu), nhtan@cpu.edu.cn (N. Tan).
These authors contribute equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113576
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 1. Structural characteristic of NF-kB inhibitors 1e7 summarized by literature investigation.
Fig. 2. Skeleton frequency analysis by the cheminformatics tools RDKit.
2

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 3. Rational drug-design of target compounds 17e19, 20e27, 28e36 and 37e40.
Fig. 2. Compared with Fig. 1, almost all the top skeletons were
similar to those of Fig. 2, especially the skeletons colored with red.
The results claimed that the skeletons colored with red might be
beneficial to the bioactivity for drug design.
2. Results and discussion
2.1. Chemistry
In our previous study, a series of natural products RAs were
found and among them RA-V can effectively inhibit NF-kB signaling
The preparations of target compounds 17e40 were described in
Scheme 1.
pathway by targeting TAK1 [15]. And the most amazing thing is that
RA-V contains the chemical characteristics and top skeletons
summarized in Figs. 1 and 2. Basing on the results summarized
above and our previous work, some interesting things could be
found that the structure features of 3-atom length linker and
phenyl ether were important to the bioactivity. Moreover, the
features of RA-V and RA-XII, which contain double ring structure,
might limit their spatial conformation to perform better bioactivity.
Because pyrimidine structure was applied in many drug-design and
contained various bioactivities such as anticancer, anti-
inflammation and antimicrob [19e21], it was introduced into the
The key intermediates 4-substituted-2-chloropyrimidines 8a-e
were synthesized from 2,4-dichloropyrimidine via substitution
with the small molecular amines a-e.
The key intermediates 12a-b were synthesized from 3-
aminothiophene-2-carboxylic acid 9a or 2-aminothiophene-3-
carboxylic acid 9b via methylation, cyclization and chlorination,
respectively. Then, the intermediates 14a-b and 14e were obtained
by substitution reaction with 4-aminphenol 13. And the in-
termediates 14c-d were synthesized from 14a-b via N-Boc protec-
tion, methylation and de-protection, respectively. Finally,
substitution and condensation of amides 14a-e with the (bromo-
methyl)benzene/4-substituted benzoic acid 15/16a-b or 4-
substituted-2-chloropyrimidine 8a-e were carried out by p-
3
-atom length linker moiety. All considerations were taken into
compounds design to yield the first and second series of com-
pounds 17e19 and 20e27. Depending on the results of the first two
series of compounds, the pyrimidine structure was remained and
the thieno[3, 2-d]pyrimidine moiety was replayed with thieno[2,3-
d]pyrimidine, which was inspired by the bioisosteres rule, to yield
the third series of compounds 28e36. Finally, decreased the steric
effects and inspired by the bioisosteres rules, the thiophene ring
was opened to obtain the fourth series of compounds 37e40.
Compared with the first three series of compounds, the fourth se-
ries showed better anticancer activity, which revealed that the N-
methylpicolinamide structure may be a privileged residue. The
design strategy for all target compounds was described in Fig. 3.
Finally, we disclosed bioactivities of all synthetic compounds
particularly N-methylpicolinamide and thienopyrimidine de-
rivatives against A549 (human lung cancer), HCT116 (human colon
cancer), MDA-MB-231 (human breast cancer) cancer cell lines, NF-
ꢀ
toluene sulfonic acid in 1,4-dioxane at 80 C to obtain the target
compounds 17e27, 28e36 and 37e40, respectively.
ꢀ
Reagents and conditions: (a) Methanol, Et
3
N, 0 C, 12 h; (b)
ꢀ
ꢀ
Methanol, SOCl
2
, 0 C, 12 h; (c) Formamidine acetate, EtOH, 110 C,
ꢀ
6 h, 90％; (d) Phosphorus oxychloride, DMF, 110 C, 3 h, 95％; (e)
ꢀ
H
2
O, sodium carbonate, sodium hydroxide, tetrahydrofuran, 80 C,
6 h; (f) Ditertbutyl pyrocarbonate, sodium bicarbonate, acetone/
ꢀ
H
CH
2
O (3:1), rt, 24 h; (g) CH
3
I, NaH, DMF, 0 C, 6 h; (h) CF
3
COOH/
ꢀ
ꢀ
2
Cl
2
, 0 C, 3 h; (i) DMF, NaHCO
3
, 40 C, 7 h; HATU, HOAT, DIPEA;
DMF, 30 C, 12 h; p-Toluene sulfonic acid, 1,4-dioxane, 80 C, 12 h.
ꢀ
ꢀ
2.2. Biological evaluation
Taking RA-V and RA-XII as reference compounds, the target
compounds 17e40 were evaluated for the cytotoxicity against three
cancer cell lines (MDA-MB-231, HCT116 and A549) by 3-(4,5-
dimethylthiazoly)-2,5-diphenyltetrazolium bromide (MTT) cell
proliferation assay. The potential compounds 22, 24, 29, 32, 36, 37,
38 and 40 were further evaluated for the activities against TAK1
k
B signaling pathway and TAK1 kinase base on our previous works
[
22e25] or other literatures [26e29]. Moreover, experiments of the
cell cycle, cell apoptosis, western bolot, docking and molecular
dynamics were presented too.
3

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Scheme 1. Synthetic route of target compounds 17e40.
kinase and NF-
k
B signaling pathway, in which 5z-7-oxozeaenol,
38's activity reach nano level with an IC50 of 0.89
A549 cell lines showed better activity than RA-XII.
Furthermore, eight selected compounds (22, 24, 29, 32, 36, 37,
38 and 40) were tested for activities against the TAK1 kinase and
mM against
RA-V, and RA-XII were selected as reference compounds. The re-
sults were summarized in Tables 1e4 and the values were the
average of at least two independent experiments.
Depending on the literature investigation and chemoinformatic
tools RDKit, and taking the conformation limitation into consider-
ation, the first two series of compounds 17e19 and 20e27 were
synthesized. And all the compounds were tested with percentage of
NF-
results indicated that four compounds showed inhibition on TAK1
with the IC50 from 0.58 to 15.73 M. In addition, the compounds
(37, 38 and 40) also performed activity against NF- B signaling
pathway with an IC50 from 0.84 ± 0.08 to 3.72 ± 0.57 M. The most
potent compound 38 showed activities of TAK1 and NF- B signaling
pathway with the IC50 of 0.58 and 0.84 M, respectively. Further-
more, these compounds, which can both inhibit the TAK1 and NF-
B signaling pathway, have provided a privileged structure for
further optimization.
kB signaling pathway, which were displayed in Table 4. The
m
k
m
k
inhibition under 20
mM as descripted in Table 1 and Fig. 4. From
Table 1 and Fig. 4, two conclusions could be summarized: 1) all the
compounds showed better selectivity toward A549 cell lines than
that of MDA-MB-231 and HCT116 cell lines; 2) compounds 20e27,
conformation limited with pyrimidine moiety, showed better
antitumour activity than compounds 17e19, which claimed that
the pyrimidine moiety was benefit to the anticancer activity.
Basing on the results of the first two series of compounds 17e19
and 20e27, it suggested that the pyrimidine moiety should be
reminded and the thieno[2, 3-d]pyrimidine moiety could be
replaced with thieno[3, 2-d]pyrimidine to investigate the effect of
anticancer activity toward compounds. As a result, compounds
m
k
2.3. Cell cycle of compound 38
To explore the cell proliferation inhibition by compound 38, cell
cycle distribution analysis was carried out on A549 cells. As
descripted in Fig. 5, compared with the control group, compound
2
8e36 were synthesized and tested with percentage inhibition
under 20 M against three cell lines as displayed in Table 2. Ac-
cording to expectations, compounds 28e36 containing thieno[2,3-
d]pyrimidine moiety (IC50: 4.02e19.57 M) performed better to-
ward A549 cell lines than that of thieno[2,3-d]pyrimidine moiety in
0e27 (IC50: 6.51e20.50 M). What's more, the amine modified
3
8 can induce cell cycle arrest of A549 cells at the G2/M checkpoint
in a dose-dependent manner. As the concentrations of compound
8 increasing, the G2/M phase population was increased from
5.91% to 30.57%, while the S phase was decreased and the G0/G1
phase was no significant change. What's more, at the concentration
of 1.8 M, compound 38 induced cell cycle arrest of A549 cells at the
G2/M checkpoint with 30.57%, which was better than that of RA-XII
25.06%). In addition, at the concentration of 1.8 M, compound 38
m
3
1
m
2
m
m
with methyl was no significant improvement toward the activity,
but even decreased. For example, compounds 28e32 showed better
anticancer activity than compounds 33e36, which is similar with
the second series of compounds 20e27.
(
m
induced cell cycle arrest of HCT116 cells at the G2/M checkpoint
with 36.72% (Fig. S1).
Moreover, the thiophene ring was open to study the steric effect
toward anticancer activity by replaced thieno[2,3-d]pyrimidine
structure with N-methylpicolinamide to yield compounds 37e40
2.4. Apoptosis of compound 38 by flow cytometry
(
Table 3). Compared with the first three series of compounds, the
In order to further reveal the mechanism of apoptosis of A549
anticancer activity of compounds 37e40 were improved obviously
with the IC50 of 3.41 ± 0.10, 0.89 ± 0.03, 7.34 ± 0.67 and
3
induced by the compound 38, the experiment of Annexin V/PI
staining was carried out (Fig. 6). Compared to the control group,
compound 38 could induce the early apoptotic significantly with a
.19 ± 0.29 mM, respectively. And the most promising compound
4

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
value of 25.40% and only 9.54% late apoptotic under the concen-
2.7. Molecular dynamics (MD) simulations
tration of 1.8 mM. And the same trend was also observed in other
concentrations. In addition, the number of apoptotic and dead cells
increased with increasing concentrations of the compound. What's
2.7.1. Molecular dynamics
In order to evaluate the system of 4GS6 in the complex with
small molecules whether stability or not, the molecular dynamics
simulations were conducted in Gromacs soft package 2019.3. The
docking poses of 29, 37, 38 and 40 with 4GS6 underwent 120 ns.
MD simulations and stable MD simulations trajectories were uti-
lized for data extracting and binding free energy calculation. The
root-mean-squared deviation (RMSD) values of the protease
backbone atoms during the MD simulation are relative to their
crystal structures. And the PCA method was used to analysis the
stable conformation, with a 2D and 3D free energy landscape
descripted in Fig. 9.
more, at the concentration of 1.8
induce the early and late apoptotic significantly with a value of
1.4% in HCT116 cell. Therefore, it can be concluded that the target
mM, compound 38 also could
7
compound 38 could induce apoptosis in
dependent manner.
a concentration-
2.5. Western blot analysis of compound 38
To further investigate the effect of compound 38 on the
expression of NF- B-associated proteins, the western blot assays
k
From Fig. 9a, the four MD simulations trajectories claimed that
the system of the four selected compounds and the protein were
stable after 120 ns MD simulation. Due to the compound 38's tra-
jectories were more stable than other three, the further 2D and 3D
free energy landscape analysis was carried out (Fig. 9bec). The
Fig. 9bec reveled that the lowest free energy conformation of the
complex, which colored with blue, have been marked with a value
of zero. What's more, in order to show the lowest free energy
conformation in a visual way, the gmx-cluster module was be used
and the result was descripted in Fig. 9d. And the residues of the
initial and end conformations of compound 38 descripted in
Fig. 9e-f, revealed that many residues around compound 38 was
nearly not change, including the residues of ARG44, ALA107,
GLY110, SER111, LYS158, PRO160, ASN161, LEU163 and CYS174.
were carried out. As shown in Fig. 7a and b in HCT116 cell lines, for
p65 and P-p65 protein, compound 38 could significantly inhibit
TNF-
with the control; For protein I
significantly inhibit TNF- -induced I
sion of P-I compared with the control; For TAK1 and P-TAK1
protein, compound 38 could inhibit TNF- -induced expression of
a
-induced P-p65 and reduce the expression of p65 compared
and P-I , compound 38 could
degradation and expres-
kB
a
kBa
a
kBa
kBa
a
TAK1 and P-TAK1 compared with the control. And the similar trend
of western blot was also observed in A549 cell lines descripted in
the Fig. 7c. These results suggested that compound 38 could inhibit
the TAK1 and NF-
.6. Molecular docking study
To explore the binding modes of target compounds with the
kB signaling pathways.
2
2.7.2. MM/PBSA analysis
active sites of TAK1, molecular docking simulation studies were
carried out by using Autodock 4.2 package and Pymol 2.4.0. And
basing on the in vitro inhibition results, we selected four com-
pounds (29, 37, 38 and 40) as the ligand example, and the structure
of TAK1 was selected as the docking model (PDB ID code: 4GS6)
Basing on the results of the RMSD and the free energy landscape
figure, the stable MD simulations trajectories were utilized for date
extracting to analysis the total binding energy. The results revealed
that the total binding free energy of 29, 37, 38 and 40
was ꢁ102.612, ꢁ95.327, ꢁ110.633 and ꢁ97.147 kcal/mol for the
complexes of 4GS6-29, 4GS6-37, 4GS6-38 and 4GS6-40, respec-
tively. And the trend shown by these data was agreed with the IC50
except compound 29 as displayed in Table 5.
[
13]. In detail, for 29 (Fig. 8a), the compound was bound to the
activity sites by two pi-alkyl bonds with CYS174 and VAL50,
respectively. Moreover, it formed with VAL42 and LYS158 by two H-
bonds to bound to the activity sites of 4GS6.
To further identify the key residues contributing to binding af-
finity, we also conducted an energy decomposition analysis. Usu-
ally, a residue, which is considered to be important for interaction
energy with ligand, is lower than ꢁ4.18 kJ/mol [30]. As shown in
Fig. 10aed, the 4GS6-38, which have maximum residues (four
residues) with a value below ꢁ4.18 kJ/mol compared with 4GS6-29
Compared with 29 (Figs. 8a), 37 (Fig. 8b) not only formed two H-
bonds interactions with residues (SER111 and GLU77), but con-
tacted with three residues (VAL42, LYS63 and LEU163) by three pi-
alkyl to further enhanced the inhibitory, which explained that
compound 37 showed a little better activity than compound 29.
In the binding mode (Fig. 8c), compound 38 was bound to the
active binding sites of 4GS6 via three hydrogen bonds and two
piealkyl interactions. In detail, the compound formed two
(one residue), 4GS6-37 (two residues) and 4GS6-40 (none residue),
have explained the reason why compound 38 showed the best
activity against TAK1. Furthermore, the residues contributing
analysis revealed that the VDW Energy played an important role for
free energies as showed in Fig. 10eef. This information may provide
a theoretical basis for further lead optimization.
piealkyl/pi-
p stacked interactions with TYR106 and VAL42,
respectively. Moreover, 4-(piperidin-1-yl)pyrimidine moiety and
N-methylpicolinamide moiety were bound to ALA107, PRO160 and
ASN161 by three H-bonds. What's more, more H-bonds than other
three compounds may explained the reason that compound 38
showed the best activity.
2.7.3. Contact frequency analysis
Compound 40 was bound to 4GS6 via two hydrogen bonds and
two piealkyl interactions (Fig. 8d), which explained that the
compound 40 showed equal activity to compound 29 and 37, but
lower activity than compound 38.
Moreover, to further identify the key residues around the li-
gands, the study of the contact frequency analysis was carried out
as descripted in Fig. 11. All the information of contact frequency is
summarized in left, and the residues contact frequency exceed 0.5
Table 1
Structures and cytotoxicity of target compounds 17e27.
a
The percentage inhibition under 20
m
M was determined at least two independent experiments.
The average values of two separated determinations.
NA: Not active (IC50 > 30 M).
b
c
m
d
Used as a positive control.
5

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
6

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
were summarized in right. Basing on the results displayed in Fig. 11,
it revealed that residues VAL-42, VAL-50, ALA-61, TYR-106, ALA-
methanol was added small molecular anime slowly and stirred for
5 h. The reaction was monitored by TLC until completed. Then the
mixture was evaporated, washed with water and filtered to afford a
white solid.
107, GLY-110, SER-111 and LEU-163 were important to the activity
of compounds. And the residues, which were occurred in the re-
sults of docking and MM/PBSA analysis, supported the conclusion
of the contact frequency analysis. Furthermore, the residues of TYR-
4.3. General procedure for the key intermediates 10a-b
106, SER-111, ASN-114 and ASP175, which was reported in recent
years, played an important role for the TAK1 activity [31,32]. Those
results indicated that the contact frequency analysis result was
reliable.
To a solution of 3-aminothiophene-2-carboxylic acid 9a or 2-
aminothiophene-3-carboxylic acid 9b (0.201 mol) in CH
3
OH
ꢀ
(
200 mL) cooled to 0 C was added excessive SOCl
2
(0.603 mol)
slowly and stirred for 1 h. Then the mixture was moved to room
temperature and stirred overnight. The reaction was monitored by
TLC until completed. Then the mixture was evaporated to afford a
gray solid.
3
. Conclusions
In summary, inspired by the cheminformatics tools RDKit and
literature investigation, four series of 24 compounds contained N-
methylpicolinamide and thienopyrimidine moiety derivatives were
designed, synthesized and evaluated for the IC50 values against
three cancer cell lines (MDA-MB-231, HCT116 and A549), TAK1
kinase and NF-kB signaling pathway. Almost all the compounds
showed the selectivity toward the A549 cell lines. Among these
4.4. General procedure for the key intermediates 11a-b
A solution of methyl 2-aminothiophene-3-carboxylate 10a or
methyl 3-aminothiophene-2-carboxylate 10b (0.1 mol), for-
mamidine acetate (0.15 mol) dissolved in ethanol (100 mL) was
ꢀ
compounds, four compounds (29, 37, 38 and 40) against A549 cell
heated to 100 C and stirred for 6 h. The reaction was monitored by
line with the IC50 values of 4.02, 3.41, 0.89 and 3.19
evaluated for the ability of against TAK1 kinase (2.52, 1.36, 0.58 and
.26 M). Moreover, three selected compounds (37, 38 and 40) were
also tested for NF- B signaling pathway inhibitory activity with the
IC50 of 3.72, 0.84 and 7.78 M, respectively. And the most promising
compound 38 can effectively inhibit the TAK1 kinase and NF-
signaling pathway with the IC50 values of 0.58 and 0.84 M,
m
M and further
TLC until completed. Finally, the solution was cooled and poured
into ice water to give a gray-white solid.
1
m
k
4.5. General procedure for the key intermediates 12a-b
m
kB
The solution of thieno[2,3-d]pyrimidin-4(3H)-one 11a or thieno
m
[3, 2-d]pyrimidin-4(3H)-one 11b (0.100 mol) in toluene (30 mL)
respectively. What's more, compound 38 can induce cell cycle ar-
rest of A549 cells at the G2/M checkpoint with 30.57% and induce
was added to phosphorus oxychloride (80 mL) and DMF (5 mL)
slowly and stirred for 30 min. Then the solution was heated to
apoptosis (34.94%) in a concentration-dependent manner at 1.8
And western blot result showed that compound 38 could inhibit
TNF- -induced phosphorylation, degradation, p65
mM.
ꢀ
1
10 C and stirred for 3 h. The reaction was monitored by TLC until
completed. The solution was cooled to room temperature and
poured into ice water slowly to obtain a white solid without further
purification.
a
Ik
B
a
IkBa
phosphorylation and TAK1 phosphorylation, and reduce the
expression of p65. Moreover, the studies of docking, molecular
dynamics, MM/PBSA method and frequency analysis theoretically
supported the conclusions of the bioevaluation.
4.6. General procedure for the key intermediates 14a-b and 14e
To a solution of 4-aminophenol (0.012 mol) and hydrogen so-
ꢀ
4
. Experimental
dium (0.013 mol) in H O cooled to 0 C was added the sodium
2
carbonate (0.014 mol) and stirred for 30 min. Then the key in-
4
.1. Chemistry
termediates 12a-c (0.011 mol) dissolved in THF (20 mL) was added
ꢀ
to the mixture slowly and heated to 80 C for 6 h. The solution was
All melting points were obtained on a Büchi Melting Point B-540
monitored by TLC until completed, then cooled to room tempera-
ture and evaporated to afford a crude product. Finally, the crude
product was purified by column chromatography on silica gel
(CH Cl /CH OH, 60:1) to give a light-yellow solid.
apparatus (Büchi Labortechnik, Flawil, Switzerland). NMR spectra
were performed using Bruker 300 or 400 MHz spectrometers
(
Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
standard. Mass spectra (MS) were taken in ESI mode on Agilent
100 LCMS (Agilent, Palo Alto, CA, USA). All the materials were
2
2
3
1
4.7. General procedure for the key intermediates 14c-d
obtained from commercial suppliers and used without purification,
unless otherwise specified. Yields were not optimized. TLC analysis
was carried out on silica gel plates GF254 (Haiyang Chemical,
Qindao, China).
To a mixture of compound 12b (0.011 mol) and sodium bicar-
ꢀ
bonate (0.023 mol) in acetone/H
2
O (1:1) cooled to 0 C was added
ditertbutyl pyrocarbonate (0.014 mol) slowly and left standing
overnight. The solution was monitored by TLC until completed and
4.2. General procedure for the key intermediates 8a-e
then extracted with EA/H
organic layer was combined and evaporated to afford yellow oil
2
O (50 mL ꢂ 3) for three times. And the
To a solution of 2,4-dichloropyrimidine and (Et) N dissolved in
3
without further purification; the solution of the yellow oil dissolved
Table 2
Structures and cytotoxicity of target compounds 28e36.
a
The percentage inhibition under 20
m
M was determined at least two independent experiments.
The average values of two separated determinations.
NA: Not active (IC50 > 30 M).
b
c
m
d
Used as a positive control.
7

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
8

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Table 3
Structures and cytotoxicity of target compounds 37e40.
a
The percentage inhibition under 20 mM was determined at least two independent experiments.
The average values of two separated determinations.
Used as a positive control.
b
c
ꢀ
Table 4
in DMF (8 mL) cooled to 0 C was added NaH and stirred for 30 min,
Inhibitory activity of TAK1 kinase and NF-
k
B signaling pathway of compounds 22,
ꢀ
then CH
3
I was added slowly keeping the 0 C. The reaction was
monitored by TLC until completed. The mixture was poured into
the ice water and extracted with EA for three times. Combining the
24, 29, 32, 36, 37, 38 and 40.
Cpd No
IC50
(m
M)a
organic layer was evaporated to afford a yellow oil. And it was
TAK1
NF-kB pathway
ꢀ
dissolved in CH
2
Cl
2
/CF
3
COOH (3:1, 10 mL) and cooled to 0 C for 3 h.
2
2
2
3
3
3
3
4
2
4
9
2
6
7
8
0
15.73
ND b
ND
c
The solution was monitored by TLC until completed. The solution
NA
2.52
6.87
12.05
1.36
0.58
1.26
ND
ND
ND
ND
was evaporated to afford a crude product and purified by column
chromatography (CH
gray power.
2
2 3
Cl /CH OH, 120:1) on silica gel to afford a
3.72 ± 0.57
0.84 ± 0.08
7.78 ± 0.89
0.065 ± 0.002
1.85 ± 0.35
ND
d
4.8. General procedure for the preparation of target compounds
17e19, 20e27, 28e36 and 37-40
RA-V
RA-XII
5
d
ND
0.009
z-7-oxozeaenold
a
b
c
The average values of two separated determinations.
ND: Not detection.
To a solution of key intermediates 14a-b, 14c-d and 14e in 1,4-
dioxane was added 4-substituted-2-chloro-pyrimidine and p-
NA: Not active (IC50 > 30
m
M).
ꢀ
toluene sulfonic acid, and heated to 80 C for 12 h. The reaction was
d
Used as a positive control.
monitored by TLC until completed. The solution was evaporated to
afford
a
crude product, which was purified by column
9

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 4. The analysis of structure and activity based the data in Table 1. a) The value of inhibitor % against three cell lines under 20 mM summarized in Table 1; b) The average value of
inhibitor % of different kind of linkers against A549 cell line.
Fig. 5. Cell cycle progression analyses of A549 cells treated with compound 38 and RA-XII.
chromatography (CH
target compounds 17e19, 20e27, 28e36 and 37e40.
2
Cl
2
/CH
3
OH, 100:1) on silica gel to afford the
21, 23), 7.28e7.20 (m, 1H, 22), 6.97 (d, J ¼ 9.0 Hz, 2H, 12, 16), 6.62 (d,
J ¼ 8.7 Hz, 2H, 13, 15), 6.35 (t, J ¼ 6.1 Hz, 1H, 17), 4.28 (d, J ¼ 6.0 Hz,
H, 18).
2
4.8.1. N-benzyl-4-(thieno[3, 2-d]pyrimidin-4-yloxy)aniline (17)
This compound was obtained as yellow solid in 43.6% yield;
4.8.2. N-4-(dimethylamino)- (4-(thieno[3, 2-d]pyrimidin-4-yloxy)
phenyl)benzamide (18)
ꢀ
þ
1
M.P.: 125.6e127.3 C; ESI-MS m/z: 334.09[MþH] .
H
NMR
8.57 (s, 1H, 3), 7.91 (d, J ¼ 5.8 Hz, 1H, 8), 7.58
d, J ¼ 6.0 Hz,1H, 7), 7.42e7.38 (m, 2H, 20, 24), 7.34 (t, J ¼ 7.6 Hz, 2H,
(
(
400 MHz, DMSO‑d
6
)
d
This compound was obtained as yellow solid in 48.7% yield;
ꢀ
þ
1
M.P.: 124.5e125.9 C; ESI-MS m/z: 391.30[MþH] .
H NMR
10

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 6. Annexin V (x-axis) versus PI (y-axis) analyses of compound 38 showed in Fig. 6a and b, and Fig. 6a was summarized by drawing Fig. 6c.
M.P.: 135.6e137.8 C; ESI-MS m/z: 391.26[MþH] . 1H NMR
ꢀ
þ
(
6
400 MHz, DMSO‑d ) d 10.02 (s, 1H, 17), 8.72 (s, 1H, 3), 8.48 (d,
J ¼ 5.4 Hz, 1H, 8), 7.88 (overlapped, 4H, 13, 15, 21, 25), 7.69 (d,
6
(400 MHz, DMSO‑d ) d 9.14 (s, 1H, 17), 8.70 (s, 1H, 3), 8.46 (d,
J ¼ 5.4 Hz, 1H, 7), 7.35e7.26 (m, 2H, 12, 16), 6.82e6.74 (m, 2H, 22,
J ¼ 5.4 Hz, 1H, 22), 7.93 (d, J ¼ 5.9 Hz, 1H, 8), 7.92e7.87 (m, 2H, 13,
15), 7.67 (d, J ¼ 5.4 Hz, 1H, 7), 7.22e7.14 (m, 2H, 12, 16), 5.96 (d,
J ¼ 5.9 Hz, 1H, 21), 3.60e3.45 (m, 4H, 25, 28), 2.01e1.90 (m, 4H, 26,
27).
13
2
1
6
4), 3.01 (s, 6H, 27, 28); C NMR (100 MHz, DMSO‑d ) d 165.69 (18),
64.31 (1), 163.55 (5), 154.65 (3), 152.90 (11), 147.35 (23), 138.10
(
(
14), 137.76 (8), 129.65 (21, 25), 124.75 (6), 122.51 (13, 15), 121.72
12, 16), 121.35 (20), 117.31 (7), 111.25 (22, 24), 40.60 (27, 28).
4.8.5. N-4-(piperidin-1-yl)- (4-(thieno[3, 2-d]pyrimidin-4-yloxy)
4.8.3. N-4-(diethylamino)- (4-(thieno[3, 2-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (21)
phenyl)benzamide (19)
This compound was obtained as yellow solid in 58.7% yield;
ꢀ
þ
1
This compound was obtained as yellow solid in 54.3% yield;
M.P.: 134.2e136.1 C; ESI-MS m/z: 405.26[MþH] .
H NMR
ꢀ
þ
1
M.P.: 131.4e133.7 C; ESI-MS m/z: 419.25[MþH] .
H
NMR
(400 MHz, DMSO‑d 9.12 (s, 1H, 17), 8.70 (s, 1H, 3), 8.46 (d,
6
) d
(
400 MHz, DMSO‑d 9.97 (s, 1H, 17), 8.72 (s, 1H, 3), 8.48 (d,
6
)
d
J ¼ 5.4 Hz, 1H, 22), 7.95 (d, J ¼ 6.0 Hz, 1H, 8), 7.85e7.78 (m, 2H, 13,
15), 7.67 (d, J ¼ 5.4 Hz, 1H, 7), 7.25e7.18 (m, 2H, 12, 16), 6.28 (d,
J ¼ 6.1 Hz, 1H, 21), 3.61 (t, J ¼ 5.4 Hz, 4H, 25, 29), 1.64 (d, J ¼ 6.0 Hz,
2H, 27), 1.54 (q, J ¼ 6.1, 4.8 Hz, 4H, 26, 28).
J ¼ 5.4 Hz, 1H, 8), 7.86 (overlapped, 4H, 13, 15, 21, 25), 7.68 (d,
J ¼ 5.4 Hz, 1H, 7), 7.34e7.25 (m, 2H, 22, 24), 6.78e6.69 (m, 2H, 12,
1
3
1
6), 3.43 (q, J ¼ 7.0 Hz, 4H, 27, 28), 1.13 (t, J ¼ 7.0 Hz, 6H, 29, 30);
C
NMR (100 MHz, DMSO‑d
54.65 (3), 150.28 (11), 147.27 (23), 138.19 (14), 137.76 (8), 129.99
21, 25), 124.75 (6), 122.50 (13, 15), 121.63 (12, 16), 120.45 (20),
17.31 (7), 110.58 (22, 24), 44.23 (27, 28), 12.86 (29, 30).
6
) d 165.70 (18), 164.32 (1), 163.55 (5),
1
(
1
4
.8.6. N-4-morpholino- (4-(thieno[3, 2-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (22)
This compound was obtained as yellow solid in 59.2% yield;
ꢀ
þ
1
M.P.: 124.2e126.7 C; ESI-MS m/z: 407.26[MþH] .
H NMR
4.8.4. N-4-(pyrrolidin-1-yl)- (4-(thieno[3, 2-d]pyrimidin-4-yloxy)
(400 MHz, DMSO‑d 9.22 (s, 1H, 17), 8.70 (s, 1H, 3), 8.45 (d,
6
) d
phenyl)pyrimidin-2-amine (20)
J ¼ 5.4 Hz, 1H, 22), 8.02 (d, J ¼ 6.0 Hz, 1H, 8), 7.82 (d, J ¼ 9.1 Hz, 2H,
This compound was obtained as yellow solid in 56.8% yield;
13, 15), 7.66 (d, J ¼ 5.5 Hz, 1H, 7), 7.22 (d, J ¼ 9.1 Hz, 2H, 12, 16), 6.28
11

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 7. a) Compound 38 inhibited the expression of NF-
kB-associated proteins. HCT116 cells were incubated with various concentrations of compound 38 for 24 h, and then treated
with 10 ng/mL TNF-a for 10 min. The cell lysates were prepared for western blot analysis with the indicated antibodies. b) The statistical analysis results of Fig. 7a were displayed in
Fig. 7b. #P < 0.05, P < 0.01 and
2
##
###
P < 0.001 vs Blank; *P < 0.05, **P < 0.01 and ***P < 0.001 vs Ctrl. c) A549 cells were incubated with various concentrations of compound 38 for
for 10 min. The cell lysates were prepared for western blot analysis with the indicated antibodies.
4 h, and then treated with 10 ng/mL TNF-
a
(
d, J ¼ 6.1 Hz, 1H, 21), 3.68 (dd, J ¼ 5.9, 3.9 Hz, 4H, 26, 28), 3.58 (t,
3.47 (s, 3H, 29), 3.37 (m, 4H, 25, 28), 1.96 (m, 4H, 26, 27); 13C NMR
(100 MHz, DMSO‑d 164.23 (1), 163.57 (18), 161.20 (20), 160.27
5),155.70 (22),154.63 (3),148.31 (11),144.10 (14),137.76 (8),127.61
J ¼ 4.9 Hz, 4H, 25, 29).
6
) d
(
4
.8.7. N-4-(3,4-dihydroisoquinolin-2(1H)-yl)- (4-(thieno[3, 2-d]
(12, 16), 124.76 (7), 122.13 (13, 15), 117.38 (6), 95.48 (21), 46.20 (25,
28), 38.08 (26, 27), 29.48 (29).
pyrimidin-4-yloxy)phenyl)pyrimidin-2-amine (23)
This compound was obtained as yellow solid in 65.2% yield;
ꢀ
þ
1
M.P.: 134.3e136.2 C; ESI-MS m/z: 453.23[MþH] .
400 MHz, DMSO‑d 9.23 (s, 1H, 17), 8.71 (s, 1H, 3), 8.46 (d,
J ¼ 5.5 Hz, 1H, 22), 8.02 (d, J ¼ 6.0 Hz, 1H, 8), 7.86 (d, J ¼ 9.1 Hz, 2H,
3, 15), 7.68 (m, 1H, 7), 7.23 (overlapped, 6H, 12, 16, 30, 31, 32, 33),
.36 (d, J ¼ 6.1 Hz, 1H, 21), 4.78 (s, 2H, 29), 3.86 (m, 2H, 25), 2.92 (t,
J ¼ 5.9, 2H, 26).
H NMR
(
6
)
d
4.8.9. N-methyl-4-(piperidin-1-yl)-N-(4-(thieno[3, 2-d]pyrimidin-
4-yloxy)phenyl)pyrimidin-2-amine (25)
1
6
This compound was obtained as yellow solid in 42.6% yield;
ꢀ
þ
1
M.P.: 126.1e128.4 C; ESI-MS m/z: 419.35[MþH] .
H NMR
(400 MHz, DMSO‑d6)
d
8.73 (s, 1H, 3), 8.48 (d, J ¼ 5.5 Hz, 1H, 22),
7
.86 (d, J ¼ 6.0 Hz,1H, 8), 7.69 (d, J ¼ 5.4 Hz,1H, 7), 7.43 (d, J ¼ 9.0 Hz,
4
4
.8.8. N-methyl-4-(pyrrolidin-1-yl)-N-(4-(thieno[3, 2-d]pyrimidin-
-yloxy)phenyl)pyrimidin-2-amine (24)
2H, 13, 15), 7.31 (d, J ¼ 8.9 Hz, 2H, 12, 16), 6.18 (d, J ¼ 6.1 Hz, 1H, 21),
3.51 (t, J ¼ 5.7 Hz, 4H, 25, 29), 3.46 (s, 3H, 30), 1.60 (m, 2H, 27), 1.49
13
This compound was obtained as yellow solid in 54.3% yield;
6
(m, 4H, 26, 28); C NMR (100 MHz, DMSO‑d ) d 164.21 (1), 163.57
ꢀ
þ
1
M.P.: 121.4e123.8 C; ESI-MS m/z: 405.35[MþH] .
H
NMR
(18), 161.90 (20), 161.31 (5), 156.61 (22), 154.61 (3), 148.40 (11),
144.05 (14), 137.75 (8), 127.69 (12, 16), 124.76 (7), 122.20 (13, 15),
117.37 (6), 94.67 (21), 44.84 (25, 29), 38.15 (26, 28), 25.58 (27), 24.73
(30).
(
400 MHz, DMSO‑d
6
) d
8.73 (s, 1H, 3), 8.48 (d, J ¼ 5.4 Hz, 1H, 22),
7
2
.84 (d, J ¼ 5.9 Hz,1H, 8), 7.69 (d, J ¼ 5.4 Hz,1H, 7), 7.45 (d, J ¼ 8.8 Hz,
H, 13, 15), 7.30 (d, J ¼ 8.8 Hz, 2H, 12, 16), 5.89 (d, J ¼ 5.8 Hz, 1H, 21),
12

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 8. The 3D interaction map between the ligands (29 (Figs. 8a), 37 (Fig. 8b), 38 (Figs. 8c) and 40 (Fig. 8d) and the protein (4GS6), the hydrogen bonds and the pi-alkyl bonds were
colored with red and gray, respectively.
4
.8.10. N-methyl-4-(indolin-1-yl)-N-(4-(thieno[3, 2-d]pyrimidin-4-
4.8.13. N-4-(piperidin-1-yl)-(4-(thieno[2, 3-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (29)
yloxy)phenyl)pyrimidin-2-amine (26)
This compound was obtained as yellow solid in 62.3% yield;
This compound was obtained as yellow solid in 54.3% yield;
ꢀ
þ
1
ꢀ
þ
1
M.P.: 132.5e134.6 C; ESI-MS m/z: 453.28[MþH] .
H
NMR
8.75 (s, 1H, 3), 8.50 (d, J ¼ 5.4 Hz, 1H, 22),
.13 (d, J ¼ 5.9 Hz,1H, 8), 7.83 (d, J ¼ 8.1 Hz,1H, 7), 7.71 (d, J ¼ 5.4 Hz,
H, 31), 7.47 (m, 2H,13,15), 7.40 (m, 2H,12,16), 7.17 (m,1H, 29), 7.00
M.P.: 126.4e128.2 C; ESI-MS m/z: 405.35[MþH] .
H NMR
(
8
1
400 MHz, DMSO‑d
6
)
d
(400 MHz, DMSO‑d 9.10 (s, 1H, 17), 8.62 (s, 1H, 3), 7.95 (over-
6
) d
lapped, 2H, 8, 22), 7.81 (d, J ¼ 8.9 Hz, 2H, 13, 15), 7.65 (d, J ¼ 5.9 Hz,
1H, 9), 7.18 (d, J ¼ 9.0 Hz, 2H, 12, 16), 6.27 (d, J ¼ 6.1 Hz, 1H, 21), 3.61
(
m, 1H, 30), 6.86 (m, 1H, 32), 6.18 (d, J ¼ 6.0 Hz, 1H, 21), 3.97 (t,
(t, J ¼ 5.5 Hz, 4H, 25, 29), 1.64 (d, J ¼ 5.3 Hz, 2H, 27), 1.57e1.50 (m,
1
3
13
J ¼ 8.7 Hz, 2H, 25), 3.53 (s, 3H, 33), 3.16 (t, J ¼ 8.7 Hz, 2H, 26);
C
4H, 26, 28); C NMR (100 MHz, DMSO‑d
6
)
d
169.49 (5), 163.78 (1),
NMR (100 MHz, DMSO‑d
6
)
d
164.17 (1), 163.61 (18), 161.61 (5),
161.93 (20), 161.38 (18), 156.68 (22), 153.47 (11), 148.74 (3), 143.90
(14), 127.84 (8), 127.73 (12, 16), 122.16 (13, 15), 119.12 (9), 119.02 (6),
94.66 (21), 44.85 (25, 29), 38.18 (26, 28), 25.59 (27).
1
(
1
59.24 (20), 157.35 (22), 154.58 (3), 149.43 (11), 144.10 (28), 143.63
14), 137.79 (27), 132.33 (8), 128.90 (12, 16), 127.27 (31), 124.97 (7),
24.80 (29), 122.82 (13, 15), 122.01 (30), 117.50 (6), 116.41 (32),
9
6.69 (21), 48.39 (25), 38.76 (33), 27.30 (26)
4.8.14. N-4-morpholino-(4-(thieno[2, 3-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (30)
4
.8.11. N-methyl-4-(3,4-dihydroisoquinolin-2(1H)-yl)-N-(4-(thieno
This compound was obtained as yellow solid in 53.4% yield;
[3, 2-d]pyrimidin-4-yloxy)phenyl)pyrimidin-2-amine (27)
ꢀ
þ
1
M.P.: 121.8e123.4 C; ESI-MS m/z: 407.31[MþH] .
400 MHz, DMSO‑d 9.20 (s, 1H, 17), 8.61 (s, 1H, 3), 8.02 (d,
J ¼ 6.0 Hz, 1H, 22), 7.96 (d, J ¼ 6.0 Hz, 1H, 8), 7.80 (d, J ¼ 9.0 Hz, 2H,
3, 15), 7.65 (d, J ¼ 5.9 Hz, 1H, 9), 7.19 (d, J ¼ 9.0 Hz, 2H, 12, 16), 6.29
d, J ¼ 6.1 Hz, 1H, 21), 3.68 (dd, J ¼ 5.7, 3.9 Hz, 4H, 25, 29), 3.58 (t,
H NMR
This compound was obtained as yellow solid in 64.2% yield;
(
6
) d
ꢀ
þ
1
M.P.: 137.3e139.1 C; ESI-MS m/z: 467.37[MþH] .
400 MHz, DMSO‑d
8.74 (s, 1H, 3), 8.49 (dd, J ¼ 7.0, 5.3 Hz, 1H,
2), 7.93 (d, J ¼ 6.0 Hz, 1H, 8), 7.70 (d, J ¼ 5.5 Hz, 1H, 7), 7.46 (d,
J ¼ 8.9 Hz, 2H, 13, 15), 7.34 (d, J ¼ 8.9 Hz, 2H, 12, 16), 7.19 (m, 4H, 30,
1, 32, 33), 6.28 (d, J ¼ 6.1 Hz, 1H, 21), 4.69 (s, 2H, 29), 3.78 (t,
J ¼ 5.8 Hz, 2H, 25), 3.50 (s, 3H, 34), 2.87 (t, J ¼ 6.0 Hz, 2H, 26).
H NMR
(
2
6
) d
1
(
13
J ¼ 4.9 Hz, 4H, 26, 28); C NMR (100 MHz, DMSO‑d
64.00 (1), 162.78 (20), 159.86 (18), 157.09 (22), 153.50 (11), 146.00
3),139.31 (14),127.68 (8), 122.21 (13,15),120.00 (12,16),119.06 (9),
19.00 (6), 95.73 (21), 66.31 (26, 28), 44.40 (25, 29).
6
) d 169.37 (5),
3
1
(
1
4
.8.12. N-4-(pyrrolidin-1-yl)-(4-(thieno[2, 3-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (28)
This compound was obtained as yellow solid in 65.3% yield;
4.8.15. N-4-(indolin-1-yl)-(4-(thieno[2, 3-d]pyrimidin-4-yloxy)
phenyl)pyrimidin-2-amine (31)
ꢀ
þ
1
M.P.: 123.5e124.9 C; ESI-MS m/z: 391.21[MþH] .
400 MHz, DMSO‑d 9.12 (s, 1H, 17), 8.61 (s, 1H, 3), 7.96 (d,
J ¼ 6.0 Hz, 1H, 22), 7.93 (d, J ¼ 5.9 Hz, 1H, 8), 7.89 (d, J ¼ 8.9 Hz, 2H,
3, 15), 7.65 (d, J ¼ 5.9 Hz, 1H, 9), 7.16 (d, J ¼ 8.9 Hz, 2H, 12, 16), 5.96
d, J ¼ 5.9 Hz, 1H, 21), 3.66e3.39 (m, 4H, 25, 28), 2.02e1.87 (m, 4H,
6, 27); 13C NMR (100 MHz, DMSO‑d
169.35 (5), 164.04 (1),
60.47 (20), 159.88 (18), 155.94 (22), 153.51 (11), 145.75 (3), 139.70
14), 127.63 (8), 122.12 (12, 16), 119.65 (13, 15), 119.05 (9), 119.01 (6),
6.27 (21), 46.50 (25, 28), 38.10(26, 27).
H NMR
(
6
)
d
This compound was obtained as yellow solid in 62.4% yield;
ꢀ
þ
1
M.P.: 137.1e139.6 C; ESI-MS m/z: 439.28[MþH] .
H NMR
1
(
2
1
(
9
(400 MHz, DMSO‑d 9.35 (s, 1H, 17), 8.64 (s, 1H, 3), 8.49 (d,
6
) d
J ¼ 8.2 Hz, 1H, 22), 8.19 (d, J ¼ 5.9 Hz, 1H, 8), 7.97 (d, J ¼ 6.0 Hz, 1H,
9), 7.87e7.81 (m, 2H, 13, 15), 7.67 (d, J ¼ 5.9 Hz, 1H, 31), 7.24
(overlapped, 3H, 12, 16, 29), 7.16 (d, J ¼ 7.8 Hz, 1H, 30), 6.94 (t,
J ¼ 7.3 Hz, 1H, 32), 6.33 (d, J ¼ 5.9 Hz, 1H, 21), 4.06 (t, J ¼ 8.6 Hz, 2H,
25), 3.21 (t, J ¼ 8.6 Hz, 2H, 26).
6
) d
13

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 9. a) The RMSD of the four selected compounds 29, 37, 38 and 40; b) The 2D energy landscape of compound 38; c) The 3D energy landscape of compound 38; d) The start
conformation and end conformation of compound 38; e) The residues of the initial conformation of compound 38; e) The residues of the end conformation of compound 38.
Table 5
Predicted free energies for binding compounds 29, 37, 38 and 40 to the protein 4GS6 by the MM/PBSA method.
Energy terms (kJ/mol)
VDW Energya
29
37
38
40
ꢁ182.043 ± 1.304
ꢁ18.678 ± 0.714
116.932 ± 1.262
ꢁ18.845 ± 0.105
ꢁ102.612 ± 1.118
ꢁ190.493 ± 1.237
2.427 ± 0.643
112.163 ± 1.197
ꢁ19.424 ± 0.079
ꢁ95.327 ± 1.089
ꢁ220.048 ± 1.047
ꢁ24.045 ± 0.513
154.569 ± 0.710
ꢁ21.105 ± 0.068
ꢁ110.633 ± 1.061
ꢁ171.034 ± 0.980
-14.315 ± 0.872
105.750 ± 2.694
ꢁ17.664 ± 0.119
ꢁ97.147 ± 2.704
b
EE Energy
Polar Energyc
d
Apolar Energy
Delta Totalg
a
Van der Waals energy.
Electrostatic energy.
Polar solvation energy.
Non-polar solvation energy.
b
c
d
g
Total binding free energy.
4
.8.16. N-4-(3,4-dihydroisoquinolin-2(1H)-yl)-(4-(thieno[2, 3-d]
J ¼ 6.0 Hz, 1H, 22), 7.97 (d, J ¼ 5.9 Hz, 1H, 8), 7.85 (d, J ¼ 9.1 Hz, 2H,
13, 15), 7.66 (d, J ¼ 5.9 Hz, 1H, 9), 7.28e7.17 (overlapped, 6H, 12, 16,
30, 31, 32, 33), 6.35 (d, J ¼ 6.1 Hz, 1H, 21), 4.78 (s, 2H, 29), 3.85 (d,
J ¼ 6.6 Hz, 2H, 25), 2.92 (t, J ¼ 5.9 Hz, 2H, 26).
pyrimidin-4-yloxy)phenyl)pyrimidin-2-amine (32)
This compound was obtained as yellow solid in 64.1% yield;
ꢀ
þ
1
M.P.: 134.3e136.7 C; ESI-MS m/z: 453.28[MþH] .
H NMR
(
400 MHz, DMSO‑d 9.21 (s, 1H, 17), 8.62 (s, 1H, 3), 8.02 (d,
6
) d
14

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
Fig. 11. Analysis of the frequency of the residues contacted to compounds (29, 37, 38
and 40) showed in Fig. 11aed, and the frequency greater than 0.5 was descripted in
Fig. 11eeh.
4
4
.8.18. N-methyl-4-(piperidin-1-yl)-N-(4-(thieno[2, 3-d]pyrimidin-
-yloxy)phenyl)pyrimidin-2-amine (34)
This compound was obtained as yellow solid in 62.4% yield;
ꢀ
þ
1
M.P.: 123.2e124.9 C; ESI-MS m/z: 419.26[MþH] .
H NMR
(400 MHz, DMSO‑d
6
)
d
8.64 (s, 1H, 3), 7.98 (d, J ¼ 6.0 Hz, 1H, 22),
Fig. 10. The energy decomposition analysis (Fig. 10aed) and the residue contribution
analysis (Fig. 10eeh) of compounds 29, 37, 38 and 40.
7.86 (d, J ¼ 6.0 Hz,1H, 8), 7.68 (d, J ¼ 6.0 Hz,1H, 9), 7.43 (d, J ¼ 8.9 Hz,
2H, 13, 15), 7.28 (d, J ¼ 8.9 Hz, 2H, 12, 16), 6.19 (d, J ¼ 6.0 Hz, 1H, 21),
3
2
1
.52 (t, J ¼ 5.6 Hz, 5H, 25, 29), 3.46 (s, 3H, 30), 1.70e1.38 (m, 4H, 26,
13
7, 28); C NMR (100 MHz, DMSO‑d
61.92 (20), 161.37 (18), 156.67 (22), 153.46 (11), 148.72 (3), 143.88
14), 127.83 (8), 127.72 (12, 16), 122.15 (13, 15), 119.10 (9), 119.00 (6),
6
) d 169.48 (5), 163.76 (1),
4.8.17. N-methyl-4-(pyrrolidin-1-yl)-N-(4-(thieno[2, 3-d]
pyrimidin-4-yloxy)phenyl)pyrimidin-2-amine (33)
(
This compound was obtained as yellow solid in 66.2% yield;
9
4.64 (21), 44.84 (25, 29), 38.17 (26, 28), 25.57 (27), 24.74 (30).
ꢀ
þ
1
M.P.: 124.1e126.3 C; ESI-MS m/z: 405.35[MþH] .
400 MHz, DMSO‑d
8.65 (s, 1H, 3), 7.98 (d, J ¼ 6.0 Hz, 1H, 22),
.84 (d, J ¼ 5.9 Hz, 1H, 8), 7.67 (d, J ¼ 5.9 Hz, 1H, 9), 7.44 (m, 2H, 13,
H NMR
(
7
1
3
6
) d
4.8.19. N-methyl-4-(indolin-1-yl)-N-(4-(thieno[2, 3-d]pyrimidin-4-
5), 7.27 (m, 2H, 12, 16), 5.88 (d, J ¼ 5.9 Hz, 1H, 21), 3.47 (s, 3H, 29),
yloxy)phenyl)pyrimidin-2-amine (35)
13
.36 (m, 4H, 25, 28), 1.89 (m, 4H, 26, 27); C NMR (100 MHz,
169.47 (5), 163.76 (18), 161.25 (1), 160.29 (20), 155.74
22), 153.46 (11), 148.63 (3), 143.92 (14), 127.81 (8), 127.64 (12, 16),
22.07 (13,15),119.10 (9),119.00 (6), 95.44 (21), 46.18 (25, 28), 38.10
26, 27), 25.46 (29).
This compound was obtained as yellow solid in 72.1% yield;
DMSO‑d
6
)
d
ꢀ
þ
1
M.P.: 135.4e137.6 C; ESI-MS m/z: 453.28[MþH] .
300 MHz, CDCl
8.65 (s, 1H, 3), 8.17 (d, J ¼ 6.5 Hz,1H, 22), 7.70 (d,
J ¼ 8.1 Hz, 1H, 8), 7.65e7.49 (overlapped, 2H, 9, 32), 7.48e7.33
overlapped, 4H, 13, 15, 31), 7.18 (d, J ¼ 7.3 Hz, 2H, 12, 16), 7.07 (t,
H NMR
(
1
(
(
3
) d
(
15

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
J ¼ 7.7 Hz, 1H, 30), 6.95 (td, J ¼ 7.4, 1.1 Hz, 1H, 29), 6.10 (d, J ¼ 6.5 Hz,
26), 3.07e3.01 (overlapped, 5H, 10, 27); 13C NMR (100 MHz, CDCl
166.59 (7), 164.60 (1), 161.66 (21), 156.65 (19), 152.30 (23), 151.13
(12), 149.69 (5), 148.85 (3), 136.72 (28), 134.83 (15), 132.69 (29),
28.33 (31), 127.18 (34), 126.76 (32), 126.51 (33), 121.60 (14, 16),
3
)
1
H, 21), 4.04 (t, J ¼ 8.5 Hz, 2H, 25), 3.72 (s, 3H, 33), 3.24 (t, J ¼ 8.4 Hz,
d
2
H, 26).
1
4
.8.20. N-methyl-4-(3,4-dihydroisoquinolin-2(1H)-yl)- N-(4-
121.26 (13, 17), 113.93 (2), 110.31 (6), 95.31 (22), 46.64 (30), 42.59
(26), 28.72 (27), 26.15 (10)
(
thieno[2, 3-d]pyrimidin-4-yloxy)phenyl)pyrimidin-2-amine (36)
This compound was obtained as yellow solid in 42.8% yield;
ꢀ
þ
1
M.P.: 138.4e139.8 C; ESI-MS m/z: 467.28[MþH] .
300 MHz, CDCl
8.64 (s, 1H, 3), 8.05 (d, J ¼ 6.6 Hz, 1H, 22),
.60e7.49 (overlapped, 2H, 30, 32), 7.46e7.33 (overlapped, 2H, 8, 9),
.38e7.27 (m, 2H, 13, 15), 7.25e7.10 (overlapped, 4H, 12, 16, 31, 33),
6
3
H
NMR
4.9. Methodology of RDKit tools
(
7
7
3
) d
The first step was to install the RDKit tools with anaconda (all
needed information could be found in the web of https://github.
com/rdkit/rdkit/blob/master/Docs/Book/Install.md). Next was to
create a new conda environment with RDKit. Finally the skeleton
frequency analysis could be performed in the Jupter of conda of
RDKit environment used the module of Chem of rdkit, Draw of
rdkit.Chem and MurckoScaffold of rdkit.Chem.Scaffolds. (All the
code used in this article can be found in Supporting Information).
.11 (d, J ¼ 6.6 Hz,1H, 21), 4.70 (s, 2H, 29), 3.81 (t, J ¼ 6.0 Hz, 2H, 25),
.65 (s, 3H, 34), 2.94 (t, J ¼ 6.0 Hz, 2H, 26).
4
.8.21. N-methyl-4-(4-((4-(pyrrolidin-1-yl)pyrimidin-2-yl)amino)
phenoxy)picolinamide (37)
This compound was obtained as yellow solid in 56.1% yield;
ꢀ
þ
1
M.P.: 137.2e138.4 C; ESI-MS m/z: 391.30[MþH] .
400 MHz, CDCl
8.69 (s,1H,18), 8.38 (d, J ¼ 5.6 Hz,1H, 3), 8.02 (d,
J ¼ 6.8 Hz, 1H, 23), 7.81 (d, J ¼ 6.5 Hz, 1H, 8), 7.75 (overlapped, 3H, 6,
4, 16), 7.06 (d, J ¼ 9.0 Hz, 2H, 13, 17), 6.96 (dd, J ¼ 5.6, 2.6 Hz, 1H, 2),
.92 (d, J ¼ 6.5 Hz, 1H, 22), 3.78e3.40 (m, 4H, 26, 29), 3.02 (d,
H NMR
(
3
)
d
4.10. Cytotoxicity assay in vitro
1
Cytotoxic activities of target compounds 17e40 were tested
with MDA-MB-231, HCT116 and A549 cell lines by the standard
MTT assay in vitro, with two compounds of NF-kB inhibitors RA-V
and RA-XII as positive control. Cancer cell lines were cultured in
5
13
J ¼ 5.1 Hz, 2H, 10), 2.13e2.00 (m, 4H, 27, 28); C NMR (100 MHz,
CDCl 166.60 (7), 164.61 (1), 160.02 (21), 156.06 (19), 152.27 (23),
49.66 (12),149.08 (5),148.70 (3),136.78 (15),121.25 (14,16),121.20
13, 17), 113.92 (2), 110.28 (6), 96.00 (22), 46.28 (26, 29), 26.13 (10,
3
)
d
1
(
minimum essential medium (MEM) supplemented with 10% fetal
3
bovine serum (FBS). Approximately 4 ꢂ 10 cells, suspended in
27, 28).
MEM medium, were plated onto each well of a 96-well plate and
ꢀ
incubated in 5% CO
2
at 37 C for 24 h. The tested compounds at the
4.8.22. N-methyl-4-(4-((4-(piperidin-1-yl)pyrimidin-2-yl)amino)
indicated final concentrations were added to the wells and incu-
phenoxy)picolinamide (38)
bated for 72 h. Fresh MTT was added to each well at a terminal
ꢀ
This compound was obtained as yellow solid in 57.2% yield; M.P.:
concentration of 5
mg/mL and incubated with cells at 37 C for 4 h.
ꢀ
þ 1
1
25.3e126.8 C; ESI-MS m/z: 405.35[MþH] . H NMR (400 MHz,
CDCl
8.49 (s, 1H, 18), 8.38 (d, J ¼ 5.5 Hz,1H, 3), 8.02 (d, J ¼ 5.5 Hz,
H, 23), 7.84 (d, J ¼ 6.2 Hz, 1H, 8), 7.78e7.63 (overlapped, 3H, 6, 14,
6), 7.06 (d, J ¼ 9.1 Hz, 2H,13,17), 6.95 (dt, J ¼ 5.6, 2.9 Hz,1H, 2), 6.11
The formazan crystals were dissolved in 100
mL DMSO each well,
3
)
d
and the absorbency at 492 nm (for absorbance of MTT formazan)
and 630 nm (for the reference wavelength) was measured with the
ELISA reader (InoElisaR-1). All the compounds were tested three
times in each of the cell lines. The results expressed as inhibition
rates or IC50 (half-maximal inhibitory concentration) were the av-
erages of two independent determinations and calculated by using
the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
1
1
(
2
d
d, J ¼ 6.7 Hz, 1H, 22), 3.70e3.66 (m, 4H, 26, 30), 3.02 (d, J ¼ 5.1 Hz,
13
H, 10), 1.77e1.66 (m, 6H, 27, 28, 29); C NMR (100 MHz, CDCl
3
)
166.58 (7), 164.60 (1), 161.45 (21), 156.47 (19), 152.28 (23), 150.62
(
1
12), 149.66 (5), 148.79 (3), 136.67 (15), 121.54 (14, 16), 121.22 (13,
7), 113.89 (2), 110.32 (6), 95.03 (22), 45.82 (26, 30), 26.13 (10),
2
5.63 (27, 29), 24.43 (28).
4.11. TAK1 kinase activity [33]
4
.8.23. N-methyl-4-(4-((4-morpholinopyrimidin-2-yl)amino)
The selected compounds (22, 24, 29, 32, 36, 37, 38 and 40) were
tested for their activity against TAK1 kinase using ADP-Glo™ Kinase
Assay (Progema) according to the manufacturer's instruction. 5Z-7-
oxozeaenol was prepared in DMSO and used as positive control. In
phenoxy)picolinamide (39)
This compound was obtained as yellow solid in 64.2% yield;
ꢀ
þ
1
M.P.: 124.2e125.9 C; ESI-MS m/z: 439.32[MþH] .
H
NMR
9.26 (s, 1H, 18), 8.78 (d, J ¼ 4.9 Hz, 1H, 8),
.50 (d, J ¼ 5.6 Hz, 1H, 3), 8.02 (d, J ¼ 6.0 Hz, 1H, 23), 7.88e7.81 (m,
H, 14, 16), 7.38 (d, J ¼ 2.6 Hz, 1H, 6), 7.17e7.09 (overlapped, 3H, 2,
3, 17), 6.30 (d, J ¼ 6.1 Hz, 1H, 22), 3.72e3.65 (m, 4H, 26, 30), 3.57
(
8
2
400 MHz, DMSO‑d
6
)
d
brief,1
mL diluted test compounds and 2 mL TAK1-TAB1 kinase (4 ng/
mL) were added and mixed to a white microplate, and incubated for
ꢀ
30 min at 25 C. Then, 2
m
L mixture of MBP Protein/ATP was added
ꢀ
1
to the microplate. After incubated for 2 h at 25 C, 5
reagent was added and incubated for 40 min at 25 C to label the
ADP. Finally, 10 l kinase detection reagent was added and detected
ml ADP-Glo™
13
ꢀ
(
m, 4H, 27, 29), 2.79 (d, J ¼ 4.8 Hz, 3H, 10); C NMR (100 MHz,
DMSO‑d 166.66 (7), 164.31 (1), 162.78 (21), 159.84 (19), 157.06
23), 152.86 (12), 150.77 (5), 146.97 (3), 139.52 (15), 121.48 (14, 16),
6
)
d
m
(
by Luminescence method. Percent inhibition ¼ (RLU(simple)-
120.57 (13, 17), 114.39 (2), 109.12 (6), 95.82 (22), 66.31 (27, 29),
RLU(Blank))/(RLU(Pos.Ctrl)-RLU(Blank)) ꢂ 100%.
4
4.40 (26, 30), 26.46 (10).
4.12. NF-kB signaling pathway activity assay
4.8.24. N-4-(4-((4-(3,4-dihydroisoquinolin-2(1H)-yl)pyrimidin-2-
yl)amino)phenoxy)-methylpicolinamide (40)
NF-kB inhibitory activity of compounds 37, 38 and 40 was
This compound was obtained as yellow solid in 54.2% yield;
evaluated by the NF-
RA-V and RA-XII were used as the positive control. HEK293T cells
were cultured in 24-well plates for 24 h. Using Lipofectamine 2000
k
B dependent luciferase reporter method [10],
ꢀ
þ
1
M.P.: 134.2e135.7 C; ESI-MS m/z: 453.33[MþH] .
H
NMR
(
400 MHz, CDCl
3
) d
8.45 (s, 1H, 18), 8.39 (d, J ¼ 5.6 Hz, 1H, 3),
8
1
3
2
.06e8.01 (m, 1H, 23), 7.92 (d, J ¼ 6.6 Hz, 1H, 8), 7.78 (d, J ¼ 2.5 Hz,
(Invitrogen), the 5 ꢂ
kB-luciferase reporters were transiently
H, 6), 7.71 (d, J ¼ 8.9 Hz, 2H, 14, 16), 7.27e7.16 (overlapped, 4H, 31,
2, 33, 34), 7.09 (d, J ¼ 8.9 Hz, 2H,13,17), 6.97 (dd, J ¼ 5.6, 2.6 Hz,1H,
), 6.19 (d, J ¼ 6.6 Hz, 1H, 22), 4.80 (s, 2H, 30), 3.91 (t, J ¼ 6.0 Hz, 2H,
transfected into the cells together with pTK-Renilla reporters. After
18 h, the cells were treated with various concentrations of the
selected compounds for 6 h, and then incubated with 10 ng/mL
16

L. Wang, Q. Zhang, Z. Wang et al.
European Journal of Medicinal Chemistry 223 (2021) 113576
TNF-
a
for 8 h. The luciferase activity was evaluated by the Dual
spacing in periodic boundary conditions. The prepared system was
subjected to a double minimization step: i) minimization of water
and ions constraining the solute; ii) global minimization without
any restriction. Prior to MD simulation, both systems were first
equilibrated through 0.1 ns in NVT, NPT ensemble at 300 K and
1 atm. Subsequently, 120 ns of MD production run in NPT ensemble
was carried out with the Berendsen thermostat-barostat. An inte-
gration time step of 2 fs was used. After MD simulation, 120 ns of
MD trajectory was used for analyzing MD trajectory and 2D/3D
energy landscape figure. And 2000 frames were extracted from the
20 ns (100e120ns) of trajectory for MM/PBSA analysis. The MM/
PBSA method in the Gromacs 2019.3 package was used to calculate
the binding free energies and energy decomposition [40]. The PCA
method was used to analysis the stable complex conformation with
the 2D and 3D energy landscape figure [41]. The gmx_contact
function of Gromacs 2019.3 package was used to analysis the H-
bond frequent between the ligand and 4GS6 [42].
Luciferase Reporter Assay System (Promega).
4.13. Cell cycle
5
A549 cells were seeded in a 6-well plate at 1 ꢂ 10 cells per well
and incubated for 24 h. Then cells were treated with vehicle (0.1%
DMSO) or different concentrations of compound 38 for 24 h. Cells
were harvested and fixed with ice-cold 70% ethanol at 4 C for 12 h.
Ethanol was removed and the cells were washed with cold PBS.
Then the cells were incubated in 0.5 mL of PBS containing 1 mg/mL
Rnase for 30 min at 37 C and stained with propidium iodide (PI) in
ꢀ
ꢀ
the dark for 30 min. The DNA contents were measured by flow
cytometer (BD FACSCalibur).
4.14. Cell apoptosis
After digestion, the cells were centrifuged at 1000 rpm for 5 min.
The supernatant was abandoned and the cells were collected,
gently resuspended with PBS and counted. 5e10 million resus-
pended cells were centrifuged at 1000 rpm for 5 min. Then the
Declaration of competing interest
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.
supernatant was abandoned and 195
solution was added to gently resuspend the cells. Afterwards, 5
Annexin V-FITC and 10 l of propidium iodide staining solution
ml of Annexin V-FITC binding
ml of
m
were added and the solution was mixed gently. Finally, the solution
was incubated in dark and in room temperature (20e25 C) for
Acknowledgments
ꢀ
1
0e20 min and tested by flow cytometry (BD FACSCalibur).
This work was financially supported by the National New Drug
Innovation Major Project (2017ZX09309027) of Ministry of Science
and Technology of China, the National Natural Science Foundation
of China (32070356, 31900269), and the “Double First-Class” Uni-
versity Project (CPU2018GF05) of China Pharmaceutical University.
4.15. Western bolt
The method was descripted in our previous work [15].
.16. Docking study
4
Appendix A. Supplementary data
For docking purposes, the three-dimensional structure of the
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113576.
protein TAK1 (PDB code: 4GS6) was obtained from RCSB Protein
Data Bank [34]. Hydrogen atoms were added to the structure
allowing for appropriate ionization at physiological pH and the
water was removed by the Pymol 2.4.0. The protonated state of
several important residues was adjusted and docking was carried
out by autodock 4.2 package in favor of forming reasonable
hydrogen bonds with the ligand [35]. Molecular docking of four
selected compounds into the complex structure (PDB code: 4GS6)
was carried out using the Autodock 4.2 package and analyzing the
results by the Pymol 2.4.0. Firstly, changing the ligand and protein
to pdbqt style were processed to product the gpf and dpf files.
Secondly, the gpf and dpf files were processed to product the
docking result by autogrid4 and autodock4, respectively. Thirdly,
the result was analysized by Pymol 2.4.0 soft package to displaying
the hydrogen bond, pi-pi interaction bond, surface and cartoon
model to show the relationship between the ligand and 4GS6.
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