Discovery of 5-bromo-4-phenoxy-N-phenylpyrimidin-2-amine derivatives as novel ULK1 inhibitors that block autophagy and induce apoptosis in non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejmech.2020.112782

UNC51-like kinase1 (ULK1) recruits its binding partners and initiates the autophagy process in cancer. ULK1 is significantly overexpressed in Non-small cell lung cancer (NSCLC) and negatively correlated with clinical prognosis in NSCLC patients. Based upo

Full text of DOI:10.1016/j.ejmech.2020.112782

European Journal of Medicinal Chemistry 208 (2020) 112782
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of 5-bromo-4-phenoxy-N-phenylpyrimidin-2-amine
derivatives as novel ULK1 inhibitors that block autophagy and induce
apoptosis in non-small cell lung cancer
Dejuan Sun a, Zijian Yang a, Yongqi Zhen b, Yushang Yang c, Yanmei Chen a, Yong Yuan c,
,
,
, *
Lan Zhang b **, Xiaoxi Zeng d ***, Lixia Chen a
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Wuya College of Innovation, Shenyang Pharmaceutical University,
Shenyang, 110016, China
b School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, China
c Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu, 610041, China
d West China Biomedical Big Data Center, West China Hospital, Sichuan University, Chengdu, 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
UNC51-like kinase1 (ULK1) recruits its binding partners and initiates the autophagy process in cancer.
ULK1 is significantly overexpressed in Non-small cell lung cancer (NSCLC) and negatively correlated with
clinical prognosis in NSCLC patients. Based upon the binding features of ULK1, we explored the phar-
macophore modeling to discover the common anchoring features. It was verified by synthesizing 5-
bromo-4-phenoxy-N-phenylpyrimidin-2-amine derivatives, as well as subsequently elucidating the
structure-activity relationships (SAR). Among all the obtained ULK1 inhibitors, 5-bromo-4-(2-fluoro-4-
nitrophenoxy)-N-(3,4,5-trimethoxyphenyl) pyrimidin-2-amine (3s), was the most active one. The
docking analysis was conducted to compare 3s and SBI-0206965, which further elucidated the roles of
the H-bond donor. This compound inhibited the proliferation of A549 cells and showed strong inhibitory
activity against ULK1 kinase. Moreover, we found that compound 3s could induce apoptosis while
simultaneously blocking autophagy. Collectively, these findings shed new light on compound 3s that
would be utilized as a promising candidate drug for the future NSCLC therapy.
Received 15 January 2020
Received in revised form
20 August 2020
Accepted 20 August 2020
Available online 29 August 2020
Keywords:
Non-small cell lung cancer
ULK1 inhibitors
A549 cells
Autophagy
Apoptosis
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Autophagy, a conserved biological process of phagocytosis, can
remove damaging organelles, and maintain stable and normal
Lung cancer is the low 5-year overall survival and most
frequently diagnosed cancer. Almost 2.3 million new cases of lung
cancer have been diagnosed in 2019 [1,2]. It is estimated that NSCLC
accounts for 83% of lung cancer cases. Despite significant progress
in cancer research and treatment, the 5-year survival rate of NSCLC
patients is only 15% [3]. The high incidence and morbidity rate of
lung cancer has continued to increase in the developed countries.
Consequently, it is highly efficacious to explore more efficacious
therapeutic agents to benefit patient survival and quality of life
[4,5].
physiological activities [6,7]. The autophagic process is regulated by
autophagy-related genes. Among them, autophagy-related gene 1
(Atg1), a serine/threonine protein kinase, acts as a promoter of
autophagy and regulates autophagy-initiating function [8,9].
Autophagy participates in diverse diseases, including cancer.
However, autophagy plays a dichotomous role in cancer. There is
mounting evidence that inducing autophagy could be an effective
approach to inhibit other important process in cells, such as mTOR
signaling or other stress responses [10]. The fusion peptide, Tat-
Beclin1 is reported as a specific autophagy inducer [11]. Most re-
searches reported that autophagy inhibition could suppress the
growth of advanced cancers. Hydroxychloroquine, the autophagy
inhibitor is the clinically available drug [12]. Combined hydroxy-
chloroquine with temsirolimus, doxorubicin or bortezomib in pa-
tients with refractory solid tumors, including melanoma and
glioblastoma multiforme [13,14].
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: zhanglanx_9@126.com (L. Zhang), zengxiaoxi@wchscu.cn
(X. Zeng), syzyclx@163.com (L. Chen).
https://doi.org/10.1016/j.ejmech.2020.112782
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
UNC-51 like kinase 1 (ULK1), Atg1 homologue, localizes to the
(IC50 ¼ 108 nM) and ULK2 (IC50 ¼ 711 nM). SBI-0206965 could
inhibit ULK1 phosphorylation of VPS34 to regulate autophagy and
cell survival [23].
autophagosomal membrane in mammalian cells and is diffusely
reported as the autophagic initiator [15,16]. ULK1 is component of
ULK1 complex (ULK1-ATF13-FIP200-ATG101), which plays an
important role in autophagy induction. Characterization of ULK1
expression has been identified that up-regulation of ULK1 is found
in some cancer tissues [17]. Previous study has reported that ULK1
showed up-regulated in NSCLC and negatively correlated with
prognosis in NSCLC patients. Otherwise, knockdown of ULK1
inhibited cell growth and enhanced the efficacy of cisplatin against
NSCLC cells [18]. Knockdown of ULK1 or the inhibition of ULK1 by
the selective inhibitor SBI0206965, inhibited cell proliferation,
induced cell apoptosis and enhanced the sensitivity of cisplatin
against NSCLC cells [17]. In this case, inhibition of ULK1 to regulate
autophagy process may be an efficacious therapeutic strategy.
Hitherto, multiple ULK1 inhibitors have been reported and
researched for potential cancer treatment. As shown in Fig. 1,
compound 6, a high affinity inhibitor of ULK1 (IC50 ¼ 8 nM), was
selected from a high throughput of 764 compounds with a standard
32P-ATP radioactive assay. However, compound 6 did not have
specificity for ULK1 and the usage could not be determined in
follow-up study [19]. In order to further improve the selectivity of
ULK1, the inhibitor with novel scaffold was found. Compound 3 was
less suitable as an inhibitor of ULK1, although it showed good
selectivity [20]. A TBK1 inhibitor, MRT67307 could inhibit ULK1
(IC50 ¼ 45 nM) and ULK2 (IC50 ¼ 38 nM). Its homologue MRT68921
also had a good affinity. Unfortunately, neither MRT67307 nor
MRT68921 had the specificity of ULK1. In addition, a high-
Pharmacophore modeling was well applied in the field of me-
dicinal chemistry [24,25]. Given several active molecules and
favorable interactions with the active site, ligand-based pharma-
cophore elucidated comprehensive chemical features [26,27]. To
introduce the method to understand development of ULK1-
inhibitors and explore structure-activity relationship (SAR), we
explored the pharmacophore modeling to discover the common
anchoring features. In regrading to this, we further designed and
synthesized a series of new ULK1 inhibitors according to the
structure of ULK1 inhibitors in the literature, and finally 27 new
small-molecule compounds were obtained. By studying the bio-
logical activity of these compounds, we found that 3s not only had
good inhibitory activity against tumor cells, but also showed strong
inhibitory activity against ULK1 kinase. Subsequently, we carried
out the SAR analysis and molecular docking. Further results
revealed that 3s blocked autophagy by ULK1 inhibition and induced
apoptosis in human lung carcinoma A549 cells, which would shed
new light on accelerating inhibitor discovery process of ULK1 and
exploiting potential candidate drug for the future NSCLC therapy.
2. Results
2.1. Design and chemistry
ULK1 possesses the standard kinase fold, but still exists several
unique features. The most notably feature is the large loop between
the N- and C-terminal lobes, which has substrate recognition and
kinase activity regulation. The positively charged loop extends out
from the kinase domain, which is rare in the remainder of the
kinome. Otherwise, ULK1 and ULK2 are the highly similar, while the
loop is one of the most nonconserved features [28]. The skeleton of
many inhibitors is similar to adenine, such as imatinib, infigratinib
throughput screening and structural modification revealed
a
ULK1 inhibitor having a skeleton of an anthracene derivative [21].
Compound 3g had a potent affinity for ULK1 (IC50 ¼ 45 nM), which
was consistent with MRT67307. WP1130 inhibited deubiquitinases,
resulting in an increase in ULK1 ubiquitination, thereby inhibiting
the activity of ULK1 and autophagy [22]. A FAK inhibitor, SBI-
0206965, was also an inhibitor of high selectivity for ULK1
Fig. 1. The structures of reported ULK1 inhibitors.

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
3
and lapatinib. Here we constructed the pharmacophore modeling,
which compared ligand-protein interactions and explored SAR
(Fig. 2). In the first step, we read 3D coordinates of the ULK1
inhibitors-protein complex. ULK1 inhibitors were docked into the
active site of ULK1 (PDB: 4WNO) (Fig. S1A). In order to nicely
matching molecules, an overlap factor, hydrophobicity of protein
cavities was introduced (Fig. S1B). The top1 pose was generated for
each ligand. It was important to analyze the binding characteristics
of reported ULK1 inhibitors. Firstly, based on the integrated struc-
tural features of ULK1 reported inhibitors, we found that the 2-
aminopyrimidine scaffolds exhibited typical type I interactions
with kinase hinge backbones as adenine did. Then, the flexibility of
the methionine gatekeeper exerted control over ULK1 inhibitor
binding by restricting access to the ATP-binding site deep. Addi-
tional, according to the rigidity of the chemical structure, existing
inhibitors can be divided into two categories. An aliphatic chain or
an aromatic ring occupied at the 4-position of pyrimidine. Finally, a
substituted aromatic ring located in the allosteric pocket and
occupied the hydrophobic binding site. Hydrophobic electron-
withdrawing groups in the adenine core enhanced potency. In
the second step, the pharmacophore model was wrapped into a
normalized analysis that expresses the similarity between the ULK1
inhibitors (Fig. S1C). Smaller molecules inevitably got good
matching with the pharmacophore model. The pharmacophore
characteristics of ULK1 inhibitors were analyzed preliminarily, and
we further analyzed the atomic characteristics of SBI0206965. The
structure of SBI0206965 was most consistent with the pharmaco-
phore based “fit value” (Table S1). Otherwise, as it is mentioned
above, SBI0206965 was more selective for ULK1. Consequently,
close protein atoms were identified by multiple types of ligand-
protein interactions (e.g., H-bond donors, H-bond acceptors, hy-
drophobicity, stacking, CH-p). We expanded close ligand-protein
atom to circular fragments and found ULK1 inhibitors-protein
complex fragment. To further inhibit the ULK1 binding, H-bond
donor and hydrophobic segments played an important role.
Hydrogen bond formed by H-bond donor was towards the back
pocket targeting the residues at the gatekeeper to gatekeeper þ3
position. Otherwise, the contributions of hydrophobicity also were
favorable interactions.
We examined the effect of various substituted groups and syn-
thesized a series of derivatives according to the synthetic routes
outlined in Scheme 1. The synthesis of compounds 3a-l was
introduced by using commercially available 2-hydroxybenzoic acid
as the starting material. The reaction of 2-hydroxybenzoic acid with
methylamine in ethyl acetate yielded intermediate 1. Intermediate
1 reacted with 5-bromo-2,4-dichloropyrimidine to afford inter-
mediate 2. Then it reacted with para-bromoaniline and diverse
aniline, which was prepared by refluxing reaction of THF in p-TsA,
to obtain final compounds 3a-l with 53e72% yields.
The synthetic routes of compounds 3m-u were depicted in
Scheme 2. The final compounds were yielded by two steps from
commercially available 5-bromo-2,4-dichloropyrimidine. First, the
Fig. 2. The workflow of designing ULK1 inhibitors based on pharmacophore model.

4
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
Scheme 1. Reagents and conditions: (i) Methanamine, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), ethyl acetate (EA), r.t.; (ii) 5-bromo-2,4-dichloropyrimidine, N,N-
Diisopropylethylamine (DIEA), Ethanol (EtOH), 0 ꢀC to r.t.; (iii) p-Toluenesulfonic acid (p-TsA), Tetrahydrofuran (THF), reflux.
Scheme 2. Reagents and conditions: (i) 5-Bromo-2,4-dichloropyrimidine/Dichloropyrimidine, DIEA, EtOH, 0 ꢀC to r.t.; (ii) p-TsA, N2, THF, reflux.
preparation of compounds 2m-u was employed by 5-bromo-2,4-
dichloropyrimidine and diverse phenol in ethanol. Then, com-
pounds 3m-u were synthesized by the similar procedure as com-
pounds 3a. Compound 5a was also synthesized by the similar
method as 3a.
In Scheme 3, 6a-c was prepared via 2,4-dichloropyrimidine with
N-Boc-1,3-propanediamine in isopropanol with K2CO3 at 80 ꢀC. The
desired compounds (8a-c) containing aliphatic chain were syn-
thesized using 6a-c with 3,4,5-trimethoxyaniline in the presence of
THF and p-TsA. The N-Boc group was removed by Trifluoroacetic
acid (TFA) in CH2Cl2 to provide the final compounds. Similarly, 10a-
b were carried out following reported procedures.
Bioactivities of these compounds were listed in Table 1. Firstly,
we introduced methoxy and fluorine substituents on ortho posi-
tion. Unfortunately, the synthesized compounds showed no better
activity than SBI0206965. For meta-position substituents, replace-
ment of methyl (3f), methoxy (3j) and bromine (3d) exhibited a
similar activity, whereas isopropyl substituent (3k) displayed
higher affinity for ULK1. This suggested that electronegativity of
substituents did not affect the potency for ULK1, which was
depended on the chemical space. To further explore the space, we
synthesized compound 3l, which showed no activity. For para-
position and di-substituents, these compounds displayed moder-
ate activity with no significant improvement.
To further explore the potency for ULK1, we investigated the
influence of the aryl moiety R1 and R2. Ten compounds (3m¡3u,
8a) were synthesized, and structures and bioactivities of these
compounds were shown in Table 2. According to the activity results
of 3a-3l, we chose 3-methylaniline/3,4,5-trimethoxyaniline fixed
on region I. The possible influence of various substituted was sur-
veyed in the subsequent structural modifications. We replaced N-
methylaminocarbonyl with 2-ethoxyl substituent to assess the ef-
fect of a role of chemical space, which was weak activity towards
ULK1. We investigated the effect of other electron-withdrawing
substituents. The introduction of 3-fluoro, 3-nitro, 4-bromo and
2,4-dichloro substituents did not affect the potency for ULK1, which
indicated that had no contribution to the achievement of activity.
2.2. SAR analyses of the synthesized compounds
To examine SAR, kinase inhibition assay was utilized to assess
the ability to inhibit ULK1. In the study, the following mainly three
regions were focus on discussing the bioactivities effects (Fig. S2),
including the skeleton structure, the head phenyl ether group (re-
gion I) and the terminal phenylamine group rings (region II). At
first, anilines with different substituents were investigated
(Table 1) on the bioactivity. For this purpose, we prepared a series of
the aryl moieties introducing activity. All other parts of compounds
were fixed as their original structures. A total of twelve compounds
(3a-l) were synthesized.
Scheme 3. Reagents and conditions: (i) Isopropanol, K2CO3.; reflux; (ii) 3,4,5-Trimethoxyaniline, THF, p-TsA, reflux; (iii) Trifluoroacetate, CH2Cl2, 0 ꢀC to r.t.

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
5
Table 1
SAR study on the head phenyl ether group (region I).
NO.
R
Kinase inhibitory activity (10
40.12
m
M, %)a
Anti-proliferative activity (IC50
,
m
M)b
HL60
A549
U937
MDA-MB-468
36.60 1.75
MCF-7
3a
23.46 1.75
>50
39.69 0.85
>50
3b
3c
21.56
27.42
>50
>50
>50
>50
>50
>50
>50
>50
35.46 1.87
>50
3d
3e
62.69
19.24
10.60 1.20
40.53 1.38
49.00 2.58
52.13 2.23
38.70 1.66
31.90 2.95
42.50 2.53
>50
>50
>50
3f
73.68
38.24
6.10 1.54
15.88 3.06
49.6 1.54
16.21 2.75
32.32 1.88
18.59 1.96
40.7 1.97
16.55 2.64
44.67 2.24
3g
27.33 3.12
3h
3i
17.79
38.84
38.14 0.95
28.32 2.64
>50
>50
>50
>50
45.53 3.13
43.96 1.45
32.82 2.46
57.77 1.95
3j
50.46
83.96
6.23 2.31
7.14 1.58
24.33 2.45
14.05 1.94
16.17 2.89
17.51 1.48
17.76 1.44
19.45 2.58
18.22 2.56
26.48 1.84
3k
3l
22.25
97.56
>50
>50
>50
>50
>50
SBI-0206963
6.78 1.58
25.12 0.94
18.92 1.7
18.38 0.35
26.17 0.35
a
Each compound was tested in triplicate; the data are presented as the mean SEM (n ¼ 3).
b
IC50 values obtained with cell viability assay for 24 h.
The results revealed that the electron-withdrawing group at 2, 3 or
the roles of 5-bromo. From Table 3, we found the electron-
withdrawing substituent at 5 position showed the highest activ-
ity. In addition, considering the effect of alkylamines chains on the
region II, we further designed compounds 8b-c, 10a-b. Among
them, the best activity was 5a, but obviously weaker than 3s.
Based on the above data, we could conclude that hydrophobic
interaction was more favorable on region I. Of special note was that
4 position showed poor activity. However, 3s presented a signifi-
cant improvement of activity, owing to the 4-nitro substituent,
which could interact with ULK1 closely. Otherwise, we designed to
retain aliphatic amine to meet the flexible need.
To examine the possible influence of electronegativity of skel-
eton, different substituted pyrimidines were designed to explore

6
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
Table 2
SAR study on the terminal phenylamine group rings (region II).
NO.
R1
R2
Kinase inhibitory activity (10
75.68
m
M, %)
Anti-proliferative activity (IC50, mM)
A549
U937
HL60
MDA-MB-468
31.40 0.72
MCF-7
3m
20.31 1.06
38.64 0.95
34.73 0.73
39.11 1.32
3n
3o
49.26
3.78
13.90 2.49
43.94 1.49
19.36 3.28
15.47 2.59
29.21 3.72
26.80 2.49
>50
>50
>50
>50
3p
3q
3r
40.24
25.68
16.34
24.63 2.47
10.30 0.34
48.73 0.34
51.23 3.75
18.64 2.48
>50
38.66 1.48
14.73 1.58
>50
36.68 1.38
31.32 3.85
>50
>50
19.11 2.5
>50
3s
99.15
1.94 2.35
12.92 1.49
10.89 0.98
16.83 0.86
19.60 2.38
3t
15.67
12.46
60.86
37.55 1.44
51.03 1.64
13.34 1.44
43.09 1.53
>50
>50
>50
3u
8a
>50
>50
>50
>50
20.65 1.24
21.47 2.15
33.12 2.35
21.91 2.15
SBI-0206963
97.56
6.78 1.58
25.12 0.94
18.92 1.7
18.38 0.35
26.17 0.35
3s showed the highest activity and had higher affinity for ULK1
than SBI0206965, also the cellular potency of 3s was 3e4 higher.
This compound contained a 2-fluoro-4-nitrophenyl ether group at
the region II position and 3,4,5-trioxyl group at the region I
position.
with IC50 < 20
activity with IC50 ranged between 37.55 and 89.12
m
M, while homologue 3t was weaken anticancer
m
M. Generally,
compounds bearing ortho-substituents on region I 3c and 3e were
devoid of anticancer activity. Meanwhile, the congeners having
meta-substituents 3d and 3f exhibited good anticancer effects
(Fig. 3A). The inhibitory activities of 3a-3u against ULK1 were
evaluated. For comparison, SBI0206965, the reported ULK1 inhib-
itor, was used as standard drug. The activity for the inhibition of
ULK1 was summarized. The result showed that 3s exhibited sig-
nificant inhibitory activities on ULK1 (Fig. 3B). To further confirm
the target selectivity of 3s, a KINOME scan panel of 100 kinases
binding assays was performed by Eurofins Pharma Discovery Ser-
vices UK Limited. The results showed that 3s prominently inhibited
the kinase activity of ULK1, but still inhibited a number of kinases.
2.3. Identification of 3s as a new ULK1 inhibitor
The antiproliferative activity of synthesis compounds was pre-
sented in supporting Table 1. This study was performed on several
human cancer cell lines A549, U937, HL60, MDA-MB-468 and MCF-
7, which were derived from human lung, lymphoma, breast cancer
and acute myeloid leukemia, respectively using MTT assay. The 3s
exhibited the highest cytotoxic effect against all the tested cell lines

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
7
Table 3
SAR study on the skeleton and alkylamines chains.
NO.
R1
Br
R2
Kinase inhibitory activity (10
99.15
m
M, %)
Anti-proliferative activity (IC50, mM)
A549
U937
HL60
MDA-MB-468
16.83 0.86
MCF-7
3s
1.94 2.35
12.92 1.49
23.12 2.75
>50
10.89 0.98
19.60 2.38
5a
8b
H
H
66.96
17.66
10.08 2.54
29.65 1.71
16.21 2.67
20.37 1.76
45.78 2.12
23.55 2.24
>50
>50
10a
8c
H
I
13.41
32.31
46.39 2.79
39.64 2.45
>50
>50
>50
>50
>50
>50
39.93 2.74
43.41 3.63
10b
I
44.86
16.89 1.29
21.96 3.27
21.57 3.34
32.27 3.24
32.69 1.29
Most notably, AMPK
a
1 and FAK were still targeted (Fig. 3C,
to the ATP-binding site deep and was beneficial to improve the
activity of ulk1 inhibitor. The results showed that H-bond donor
and hydrophobic segments played an important role.
Table S2). Furthermore, to confirm whether 3s could inhibit the
kinase activity of ULK1 in cells, we detect the cellular phosphory-
lated levels of ULK1 substrates such as Atg13 (Ser318) and Beclin1
(Ser15) under normal or nutrient starvation conditions. The results
showed 3s can inhibit ULK1 activity in A549 cells, as indicated by
the decreased levels of p-Atg13Ser318 and p-Beclin1Ser15 (Fig. 3D).
2.5. 3s blocks autophagy by ULK1 inhibition in A549 cells
ULK1 is closely related to early autophagosome formation, and
phosphorylates beclin1 to trigger the autophagy cascade. So,
inhibiting ULK1 possibly blocks autophagy. We detected the ability
of 3s to inhibit autophagy and cell survival in A549 lung cancer
cells. The suppression of ULK1 expression was surveyed by
immunoblotting. Firstly, we found that 3s significantly down-
regulated the activity of ULK1 and induced downregulation of p-
ULK1 (ser317) in a concentration-dependent manner (Fig. 5A).
AMPK can directly activate ULK1 by phosphorylation at Ser317. So,
the expression of ULK1 may be decreasing due to 3s inhibition of
AMPK. We also revealed that the LC3 conversion was significantly
delayed in A549 cells. To confirm the result, autophagy flux was
researched by p62 degradation assay. Treated with 3s, p62 degra-
dation was not observed in A549 cells, and the expression of
beclin1 was decreased. As expected, treatment with 3s resulted in a
prominent inhibition of autophagy, as evidenced by an increase in
the autophagy substrate p62, a reduction in LC3 I conversion to LC3
II, and a decrease in the levels of beclin1 (Fig. 5B). Taken together,
the results suggested that inhibition of ULK1 kinase activity by 3s
blocked autophagy of A549 cells. To further assess autophagy, we
looked at the autophagy flux by GFP-mRFP-LC3 (Fig. 5C). These
results suggest that 3s can block autophagy via inhibiting ULK.
2.4. Molecular docking
Molecular docking (MD) is a widely used approach to predict the
predominant binding mode and screen potential active structures
[29]. To explore structural information of ULK1 inhibitor binding
modes, two kinase-inhibitor complexes were analyzed by MD. In
the study, the crystal structure of ULK 1 kinase domain was ob-
tained from the RCSB PDB database (http://www.rcsb.org, PDB
entry: 4WNO, 1.56 Å resolution). Compound 3s was selected as a
potential ULK1 inhibitor, based upon the consideration of kinase
inhibitory effect and anti-tumor activity. In order to investigate the
interaction of 3s with ULK1 and understand the conformation of 3s
in the active site, the analysis was conducted to compare 3s and
SBI-0206965. In this study, ligands with the most favorable binding
free energies and reasonable orientations were chosen as the
optimal docked conformation. We investigated that 3s and SBI-
0206965 could bind to ULK1 stably (Figs. S3A and B). Two in-
hibitors formed canonical hydrogen bonds with the hinge back-
bones. However, small differences were found in their binding
positions. 2-Aminopyrimidine NHs of 3s formed hydrogen bonds
with backbone carbonyl (Cys95, the gatekeeper þ3 position res-
idue) of the ULK1 hinge region, while 2-aminopyrimidine moiety
acted as an adenosine mimic, which was stronger interactions than
SBI0206965. In addition, interactions between the nitro moiety on
the region II and the backbone carbonyl of Gln142 also contributed
to strong binding. Furthermore, the pocket formed by Ile22, Val30,
Ala44 formed hydrophobic interactions with the region I group
(Fig. 4). H-bond formed at gatekeeper þ3 position restricted access
2.6. 3s promotes apoptosis in A549 cells
ULK1 is closely to elucidate the mechanisms of 3s -inhibited
A549 cells growth, the morphologic changes were observed under
inverted microscopy by Hoechst 33,258 staining. When cells were
treated with 3s, the apoptotic DNA fragments significantly
increased in dose department manner (Fig. 6A). Subsequently,

8
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
Fig. 3. Identification of 3s as the ULK1 inhibitor. (A) Treatment with a series of compounds after 24 h, anti-tumor viabilities were detected by MTT assay. (B) The kinase inhibitory
activity of ULK1 for synthesized compounds and SBI020695 were detected by kinase assay at 10 M. Kinases whose binding was inhibited by 3s were marked with red circles, and
the circle size indicates the degree of binding affinity. (C) Kinome tree represented the selectivity of 3s. The activity of kinases was measured in the presence of 10 M 3s. (D)
A549 cells were treated with 3s, under normal or nutrient starvation conditions. After treatment, the expression levels of Beclin 1, p-Beclin1Ser15, Atg13, p-Atg13Ser318 were analyzed
by immunoblotting, -actin was used as a loading control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
m
m
b
Western blot analysis revealed that bcl-2 expression was remark-
ably down-regulated, bax and the active form of caspase-3
expression were increased after 3s treatment (Fig. 6B). Taken
together, these results indicated that 3s could induce apoptosis via
the mitochondrial pathways in A549 cells.
the common anchoring features, which derived the inhibitor dis-
covery process of ULK1, including: (i) the identification of common
anchoring features of the ULK1-inhibitors interactions; (ii) the
analysis of the influence of chemical features and the interpretation
of SAR by focusing on structural modifications; (iii) the favorable
interactions of improving the true odds of high-throughput virtual
screening. Subsequently, the experiments were performed on
compound synthesis, screening, and mechanism of action. The re-
sults indicate that compound 3s displays a remarkable inhibitory
activity against ULK1, but still targeted other kinases, including FAK,
3. Conclusions
ULK1, a well-known autophagy initiator, has been reported to be
significantly up-regulated in NSCLC and negatively correlated with
clinical prognosis of patients. Consequently, discovery of new ULK1
inhibitors will be a promising strategy for the current NSCLC
treatment. We explored the pharmacophore modeling to discover
Src, JAK3 and AMPKa1. Otherwise, compound 3s inhibits the pro-
liferation of A549 cells in a concentration-dependent manner,
which is much stronger than a known ULK1 inhibitor SBI0206965.

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
9
Fig. 4. Predicted binding mode of compound 3s in the active of ULK1 (PDB: 4WNO2).
Fig. 5. Compound 3s inhibits autophagy via the ULK3s blocks autophagy by ULK1 inhibition in A549 cells. (A) A549 cells were treated with control or 3s for 24 h, and then the
expression of ULK1, p-ULK1 (ser317) was detected by Western blot analysis. (B) The expression levels of p62, Beclin 1 and LC3I/II were determined by Western blot analysis. (C) The
expression of LC3B puncta was observed by fluorescence microscope. Blue: DAPI. Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)

10
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
Fig. 6. 3s promotes apoptosis in A549 cells. (A) After treatment with Compound 3s, A549 cells were stained with Hoechst 33258. Scale bar: 20 mm. (B) After 3s treatment, the
expression levels of Bax, Bcl-2, cleaved-Caspase 3 and Caspase3 were determined by Western blot analysis.
Moreover, we further demonstrate that compound 3s can suppress
cytoprotective autophagy and promote apoptosis in NSCLC cells.
Taken together, these findings shed new light on compound 3s that
would be further utilized as a promising candidate drug for the
future NSCLC therapy.
5.5 ꢁ 104 cells/mL. After 24 h incubation, cells were treated with
different concentrations of synthetic compounds for the indicated
time periods. Cell viability was measured by the MTT assay.
4.3. Molecular docking and pharmacophore modeling
4. Experimental section
Discovery Studio software (version 3.1) was used to run all the
calculations [30]. Docking program adopts the CHARMm force field
to minimize energy of the ligands. The crystal structure of the ULK1
kinase (PDB code: 4WNO) was taken from PDB database. The
binding sphere was primarily defined within 10 Å from its original
ligand. Gold score was selected as the scoring function. Identifica-
tion of the critical pharmacophoric features is important for potent
ULK1 inhibitors. All the training set molecules were subjected to
find the common chemical features of reported ULK1 inhibitors.
Hydrogen bond acceptor, hydrogen bond donor, ring aromatic and
hydrophobic aromatic chemical features were selected with the
parameters of minimum ‘‘000 to maximum of ‘‘5’’. Ten quantitative
pharmacophore models were generated with corresponding sta-
tistical parameters such as cost values, root mean square and fit
values. The best quality hypothesis was selected. Images were
created using PyMOL.
4.1. Cell culture and reagent
All the cell lines used in this study were purchased from Amer-
icanType Culture Collection (ATCC, Manassas, VA, USA). A549, MDA-
MB-468 and MCF-7 cells were cultured in Dulbecco Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum and
incubated with 5% CO2. HL60 and U937 cells were cultured in RPMI
1640 culture medium supplemented with 10% fetal bovine serum.
MTT (#M2128) was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Primary antibodies against ULK1 (#8054), p-ULK1ser317
(#12753), p62 (#8025), Beclin 1 (#3495), p-Beclin1ser15 (#84966),
Bax (#5023), Bcl-2 (#2870), Caspase-3 (#9662), Atg13 (#13468), p-
Atg13ser318 (#46329) were purchased from Cell Signaling Tech-
nologies (Boston, MA, USA). Rapamycin (#ab120224) was purchased
from Abcam (Cambridge, UK). GFP/mRFP-LC3 (HB-AP2100001) was
purchased from HANBIO (China). ADP-Glo™ Kinase Assay (#V9101)
and ULK1 Kinase Enzyme System (#V3521) were purchase from
Promega (Madison, WI, USA).
4.4. Hoechst 33258 staining
Cells (1 ꢁ 105 per well) were treated Compound 3s or rapamycin
together with Compound 3s for 24 h. After washed twice with cold
PBS, the cells were stained with Hoechst 33258 in the dark at 37 C
for 30 min. Then the morphological change of cell was observed
under fluorescence microscope.
4.2. Cell viability assay
Cells were dispensed in 96-well plates at
a density of

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
11
4.5. GFP/mRFP - LC3 transfection
independent experiments. The data are expressed as means SD
and analyzed with GraphPad Prism 6.0. Statistical comparisons
were made by 1-way ANOVA and Student’s t-test of SPSS 17.0
(Chicago, IL, USA). P < 0.05 was considered statistically significant.
The method was as previous description [31,32]. The A549 cells
were sequentially incubated, starting with GFP/mRFP-LC3 trans-
fected for 6 h. Then the transfected cells were used for subsequent
experiments 24 h later and were analyzed under a fluorescence
microscope.
4.10. General procedure for the synthesis of compounds 3a-3u, 5a,
8a-c, 10a-b
4.6. Flow cytometric analysis
To a solution of 2-hydroxybenzoic acid (30.0 mmol), and EDCI
(30.0 mmol) in ethyl acetate (250 mL) was added methylamine
(150.0 mmol) after stirring for 10 min at room temperature. The
reaction was allowed to r.t. for overnight. After completion, the
solvent was removed under reduced pressure to give the crude
product. The crude product was purified by silica gel flash chro-
matography (petroleum ether/ethyl acetate 5:1) to afforded inter-
mediate 1a. A 100 ml round bottom flask was charged with 50 ml
ethanol at 0 ꢀC. After 15 min, the intermediate 1, 5-bromo-2,4-
dichloropyrimidine and DIEA were added dropwise at 0 ꢀC, and
removed to room temperature for additional 8 h. Upon completion,
the residue was concentrated to give the crude oil. Intermediate 2a
was purified by silica gel flash chromatography (petroleum ether/
ethyl acetate 3:1) as a colorless oil. The mixture of 2a (0.5 mmol, 1
eq), 3,4,5-trimethoxyaniline (0.6 mmol, 1.2 eq) and p-TsA
(0.1 mmol, 0.2 eq) in THF was stirred at 80 ꢀC overnight. After the
completion of the reaction, the crude product was purified by silica
gel using PE/EA (3:1) to produce 3a. The procedure was also applied
to the following compounds 3b-u, 5a. To a solution of 2,4-
dichloropyrimidine (10 mmol), N-Boc-1,3-propanediamine
(10 mmol) and K2CO3 (20 mmol) were stirred in isopropanol and
heated at 80 ꢀC for 24 h. After the completion of the reaction, the
filtration was processed to give intermediate 6a. The mixture of 6a
(1 mmol,1 eq), 3,4,5-trimethoxyaniline (1.2 mmol,1.2 eq) and p-TsA
(0.2 mmol, 0.2 eq) in THF was stirred at 80 ꢀC overnight. The re-
action mixture was subsequently evaporated, and the residue was
purified by chromatography (petroleum ether/ethyl acetate ¼ 4:1)
to 7a. The N-Boc group was removed by TFA in CH2Cl2 to provide
the final compound 8a. Similarly, 10a-b were yielded by 9a-b with
N, N0-diethyl-ethylenediamine.
For Annexin V-FITC/PI staining, the treated cells were collected,
washed twice with PBS and then stained with Annexin V-FITC
(1:1000) in binding buffer at room temperature in the dark. 15 min
later, the cells were incubated with PI staining solution for 5 min.
Then the cells were measured by flow cytometry (Becton
Dickinson).
4.7. Western blot analysis
Western blot analysis was carried out by the method as previous
description [33]. Cells were treated with Compound 3s and
SBI0206965 for indicated times. Then both adherent and floating
cells were collected, which was added with a Rapa Buffer with
Protease Inhibitor and Phosphatase inhibitor Cocktail (Sigma-
Aldrich) after centrifugation (12,000 g at 4 ꢀC for 15 min); the
protein concentration was determined using the BCA method.
Proteins were separated by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis and transferred to nitrocellu-
lose membranes. The membranes were blocked with Tris Buffered
Saline with Tween-20 (TBST) containing 5% skimmed milk at room
temperature for 1 h, incubated overnight with primary antibodies
at 4 ꢀC and subsequently incubated with secondary horseradish
peroxidaseconjugated, goat anti-rabbit or goat anti-mouse IgG
(Abcam) at room temperature for 1 h, then visualized by using ECL
reagents.
4.8. In vitro kinase activity assays
Kinase inhibitor specificity profiling assays were first carried out
using the Kinase Profiler service of Eurofins Pharma Discovery
Services UK Limited according to the protocols described below (for
details on more kinases, see http://www.eurofins.com/
pharmadiscovery). The concentration of the compound in each
4.10.1. Preparation of 2-((5-bromo-2-((4-bromophenyl)amino)
pyrimidin-4-yl)oxy)-N-methylbenzamide (3a)
4.10.1.1. 2-((5-Bromo-2-((4-bromophenyl)amino)pyrimidin-4-yl)
oxy)-N-methylbenzamide (3a). White powder; Yield 82%; m.p.
assay was 10
mM.
212e215 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.81 (s, 1H), 8.52 (s,
ULK1 kinase activity assay was performed using ADP-Glo™ Ki-
nase Assay. The ADP-Glo™ Kinase Assay is a luminescent kinase
assay that measures ADP formed from a kinase reaction; ADP is
converted into ATP, which is converted into light by Ultra-Glo™
Luciferase. The luminescent signal positively correlates with kinase
activity. The kinase reaction is performed by kinase buffer (40 mM
Tris pH 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 50 mM DTT). The ULK1
kinase enzyme, substrate, compound, DMSO, and ATP were diluted
1H), 8.13 (d, J ¼ 4.4 Hz, 1H), 7.66 (d, J ¼ 7.6 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz,
1H), 7.45 (t, J ¼ 7.5 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.29 (d, J ¼ 7.9 Hz,
2H), 7.17 (d, J ¼ 8.5 Hz, 2H), 2.64 (d, J ¼ 4.6 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6)
d 165.84, 165.50, 160.66, 158.15, 150.05,
139.60, 131.80, 131.32 (2C), 130.10, 129.55, 126.54, 124.33, 120.99
(2C), 113.46, 93.44, 26.50; HRMS: calcd. for
C18H14Br2N4O2:
498.9381 [MþNa]þ, found: 498.9381.
in kinase buffer. Then, 1
(10 ng), or 2 l ATP (10
wells of a 384-well plate. The plate was mixed and incubated at
room temperature for 60 min. After the kinase reaction incubation,
m
l compound or DMSO, 2
m
l ULK1 kinase
4.10.1.2. 2-((5-Bromo-2-((4-(trifluoromethyl)phenyl)amino)pyr-
imidin-4-yl)oxy)-N-methylbenzamide (3b). White powder; Yield
m
m
M)/mixMBP (0.1 g/ l) were added to the
m m
72%; m.p. 229e234 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.08 (s,1H),
8.58 (s, 1H), 8.16 (d, J ¼ 4.5 Hz, 1H), 7.67 (dd, J ¼ 7.7, 1.5 Hz, 1H), 7.64
(td, J ¼ 7.6, 1.2 Hz, 1H), 7.52 (d, J ¼ 8.2 Hz, 2H), 7.46 (td, J ¼ 7.6,
0.8 Hz, 1H), 7.39 (d, J ¼ 8.1 Hz, 1H), 7.35 (d, J ¼ 8.6 Hz, 2H), 2.64 (d,
5
m
l of ADP-Glo™ reagent was added per well. Then the plates were
mixed for 2 min and incubated at room temperature for 40 min
10 l of kinase detection reagent was added to all the wells and
m
J ¼ 4.6 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 165.85, 165.61,
incubated at room temperature for 30 min. The luminescence was
measured.
160.73, 158.06, 150.06, 143.87, 131.83, 130.12, 129.57, 126.58, 126.33,
125.80, 124.33, 123.65, 121.80, 118.72 (2C), 94.29, 26.49; HRMS:
calcd. for C19H14BrF3N4O2: 489.0150 [MþNa]þ, found: 489.0151.
4.9. Statistical analysis
4.10.1.3. 2-((5-Bromo-2-((2-fluorophenyl)amino)pyrimidin-4-yl)
oxy)-N-methylbenzamide (3c). White solid; Yield 79%; m.p.
All the presented data and results were confirmed in at least 3

12
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
150e152 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.12 (s, 1H), 8.45 (s,
4.10.1.9. 2-((5-Bromo-2-((3-(trifluoromethyl)phenyl)amino)pyr-
imidin-4-yl)oxy)-N-methylbenzamide (3i). White solid; Yield 72%;
1H), 8.09 (d, J ¼ 4.5 Hz, 1H), 7.59 (dd, J ¼ 7.5, 1.3 Hz, 1H), 7.53 (td,
J ¼ 7.8, 1.5 Hz, 1H), 7.42 (td, J ¼ 8.1, 1.0 Hz, 1H), 7.39e7.30 (m, 2H),
7.18e7.08 (m, 1H), 7.03 (dd, J ¼ 12.9, 6.2 Hz, 1H), 6.90 (t, J ¼ 7.6 Hz,
m.p. 200e203 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.95 (s, 1H), 8.57
(s, 1H), 8.12 (d, J ¼ 4.4 Hz, 1H), 7.76 (s, 1H), 7.69e7.61 (m, 2H), 7.57
(td, J ¼ 7.9, 1.5 Hz, 1H), 7.40 (dd, J ¼ 15.3, 7.8 Hz, 2H), 7.24 (t,
J ¼ 7.8 Hz, 1H), 7.19 (d, J ¼ 7.6 Hz, 1H), 2.64 (d, J ¼ 4.6 Hz, 3H); 13C
1H), 2.65 (d, J ¼ 4.6 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 166.08,
165.15, 160.65, 158.89, 156.13, 153.69, 149.71, 131.52, 130.15, 129.34,
127.21, 126.30, 125.20, 124.26, 124.04, 115.75, 93.73, 26.49; HRMS:
calcd. for C18H14BrFN4O2: 439.0182 [MþNa]þ, found: 439.0181.
NMR (100 MHz, DMSO‑d6)
d 165.90, 165.46, 160.65, 158.23, 149.89,
131.75, 129.99, 129.78, 129.71, 129.49, 126.50, 125.89, 124.03, 123.18,
122.64, 118.25, 115.17, 94.25, 26.49; HRMS: calcd. for
4.10.1.4. 2-((5-Bromo-2-((3-bromophenyl)amino)pyrimidin-4-yl)
C
19H14BrF3N4O2: 489.0150 [MþNa]þ, found: 489.0151.
oxy)-N-methylbenzamide (3d). White solid; Yield 75%; m.p.
209e214 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.81 (s, 1H), 8.55 (s,
4.10.1.10. 2-((5-Bromo-2-((3-methoxyphenyl)amino)pyrimidin-4-yl)
1H), 8.11 (d, J ¼ 4.6 Hz, 1H), 7.63 (dd, J ¼ 7.6, 1.5 Hz, 2H), 7.60 (td,
J ¼ 7.8,1.6 Hz,1H), 7.41 (td, J ¼ 7.9,1.3 Hz,1H), 7.38 (d, J ¼ 8.1 Hz,1H),
7.32 (d, J ¼ 7.5 Hz, 1H), 7.07e6.93 (m, 2H), 2.64 (d, J ¼ 4.6 Hz, 3H);
oxy)-N-methylbenzamide (3j). Yellow solid; Yield 62%; m.p.
136e139 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.94 (d, J ¼ 12.5 Hz,
1H), 9.85 (s, 1H), 8.59 (s, 1H), 7.28 (s, 1H), 7.22 (dd, J ¼ 15.4, 7.6 Hz,
2H), 7.15 (t, J ¼ 8.1 Hz, 1H), 7.06 (d, J ¼ 7.7 Hz, 1H), 6.79 (t, J ¼ 7.5 Hz,
2H), 6.55 (dd, J ¼ 8.0, 1.7 Hz, 1H), 3.72 (s, 3H), 3.28 (s, 3H); 13C NMR
13C NMR (100 MHz, DMSO‑d6)
d 165.91, 165.44, 160.62, 158.18,
149.88,141.84, 131.92,130.56, 129.93, 129.54, 126.65, 124.64, 124.04,
121.86, 121.16, 117.95, 93.96, 26.50; HRMS: calcd. for
(100 MHz, DMSO‑d6) d 169.13, 161.82, 161.44, 159.95, 159.00, 155.24,
C
18H14Br2N4Oþ2 : 498.9381 [MþNa]þ, found: 498.9379.
141.19, 132.14, 129.76, 129.65, 122.77, 119.09, 116.55, 112.00, 107.75,
105.47, 104.58, 55.40, 35.75; HRMS: calcd. for C19H17BrN4O3:
451.0382 [MþNa]þ, found: 451.0376.
4.10.1.5. 2-((5-Bromo-2-((2-methoxyphenyl)amino)pyrimidin-4-yl)
oxy)-N-methylbenzamide (3e). White solid; Yield 68%; m.p.
158e160 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
8.47 (s, 1H), 8.12 (d,
4.10.1.11. 2-((5-Bromo-2-((3-isopropylphenyl)amino)pyrimidin-4-yl)
J ¼ 4.5 Hz, 1H), 8.07 (s, 1H), 7.62 (dd, J ¼ 7.6, 1.5 Hz, 1H), 7.57 (td,
J ¼ 7.8, 1.6 Hz, 1H), 7.50 (d, J ¼ 7.8 Hz, 1H), 7.40 (td, J ¼ 7.5, 0.9 Hz,
1H), 7.36 (d, J ¼ 8.1 Hz, 1H), 7.02e6.88 (m, 2H), 6.62 (t, J ¼ 5.7 Hz,
1H), 3.78 (s, 3H), 2.64 (d, J ¼ 4.6 Hz, 3H); 13C NMR (100 MHz,
oxy)-N-methylbenzamide (3k). White powder; Yield 93%; m.p.
135e137 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.55 (s, 1H), 8.49 (s,
1H), 8.08 (d, J ¼ 3.6 Hz,1H), 7.63 (d, J ¼ 7.6 Hz,1H), 7.58 (t, J ¼ 7.6 Hz,
1H), 7.39 (dd, J ¼ 17.3, 7.9 Hz, 2H), 7.22 (s,1H), 7.17 (d, J ¼ 5.4 Hz,1H),
6.94 (t, J ¼ 7.6 Hz,1H), 6.74 (d, J ¼ 7.5 Hz,1H), 2.65 (d, J ¼ 4.4 Hz, 3H),
2.59 (dd, J ¼ 13.3, 6.7 Hz, 1H), 1.08 (d, J ¼ 6.8 Hz, 6H); 13C NMR
DMSO‑d6)
d 166.06, 165.25, 160.64, 158.43, 149.81, 149.63, 131.58,
130.16, 129.40, 128.08, 126.38, 124.11, 123.61, 121.06, 120.47, 111.18,
93.36, 56.13, 26.48; HRMS: calcd. for C19H17BrN4O3: 451.0382
[MþNa]þ, found: 451.0378.
(100 MHz, DMSO‑d6)
d 165.91, 165.25, 160.55, 158.58, 150.03,
149.09, 139.99, 131.69, 130.07, 129.48, 128.59, 126.38, 124.14, 119.79,
117.56, 116.90, 92.92, 33.89, 26.51, 24.36 (2C); HRMS: calcd. for
4.10.1.6. 2-((5-Bromo-2-(m-tolylamino)pyrimidin-4-yl)oxy)-N-
C
21H21BrN4O2: 463.0746 [MþNa]þ, found: 463.0745.
methylbenzamide (3f). White solid; Yield 77%; m.p. 183e186 ꢀC; 1H
NMR (400 MHz, DMSO‑d6)
d
9.58 (s, 1H), 8.49 (s, 1H), 8.09 (d,
4.10.1.12. 2-((2-(benzo[d] [1,3]dioxol-5-ylamino)-5-bromopyrimidin-
J ¼ 4.5 Hz, 1H), 7.64 (d, J ¼ 7.6 Hz, 1H), 7.59 (td, J ¼ 7.9, 1.4 Hz, 1H),
7.41 (t, J ¼ 7.7 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.19 (s, 1H), 7.09 (d,
J ¼ 7.5 Hz,1H), 6.92 (t, J ¼ 7.8 Hz,1H), 6.66 (d, J ¼ 7.4 Hz,1H), 2.64 (d,
4-yl)oxy)-N-methylbenzamide (3l). Yellow solid; Yield 85%; m.p.
182e184 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.91 (s, 1H), 9.72 (s,
1H), 8.54 (s, 1H), 7.26e7.17 (m, 2H), 7.10 (s, 1H), 6.90e6.71 (m, 4H),
5.96 (s, 2H), 3.26 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
169.11,
J ¼ 4.6 Hz, 3H), 2.07 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
165.90,
d
165.30, 160.57, 158.54, 150.03, 140.02, 137.90, 131.73, 130.09, 129.49,
128.49, 126.44, 124.22, 122.86, 119.52, 116.53, 92.85, 26.51, 21.83;
161.82, 161.48, 158.98, 155.21, 147.44, 142.72, 134.28, 132.11, 129.68,
122.79, 119.14, 116.55, 112.61, 108.33, 103.83, 102.11, 101.30, 35.69;
HRMS: calcd. for
435.0435.
C
19H17BrN4O2: 435.0433 [MþNa]þ, found:
HRMS: calcd. for
465.0178.
C
19H15BrN4O4: 465.0174 [MþNa]þ, found:
4.10.1.7. 2-((5-Bromo-2-((2,4-dimethylphenyl)amino)pyrimidin-4-
4.10.1.13. 5-Bromo-4-(2-ethoxyphenoxy)-N-(m-tolyl)pyrimidin-2-
yl)oxy)-N-methylbenzamide (3g). White powder; Yield 70%; m.p.
amine (3m). White solid; Yield 81%; m.p. 100e103 ꢀC; 1H NMR
109e113 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
8.66 (s, 1H), 8.34 (s,
(400 MHz, DMSO‑d6)
d
9.64 (s, 1H), 8.50 (s, 1H), 7.31 (dd, J ¼ 12.3,
1H), 8.01 (d, J ¼ 4.5 Hz, 1H), 7.57 (dd, J ¼ 7.5, 1.4 Hz, 1H), 7.50 (td,
J ¼ 7.8, 1.6 Hz, 1H), 7.39e7.27 (m, 2H), 7.09 (d, J ¼ 8.1 Hz, 1H), 6.91 (s,
1H), 6.77 (d, J ¼ 7.9 Hz, 1H), 2.66 (d, J ¼ 4.6 Hz, 3H), 2.19 (s, 3H), 2.07
4.8 Hz, 1H), 7.27 (dd, J ¼ 7.9, 1.3 Hz, 1H), 7.23e7.14 (m, 2H), 7.05 (dd,
J ¼ 15.5, 7.8 Hz, 2H), 6.91 (t, J ¼ 7.8 Hz, 1H), 6.65 (d, J ¼ 7.4 Hz, 1H),
3.97 (q, J ¼ 6.9 Hz, 2H), 2.05 (s, 3H), 1.05 (t, J ¼ 6.9 Hz, 3H); 13C NMR
(s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
166.18, 164.87, 160.54,
(100 MHz, DMSO‑d6) d 165.08, 160.63, 158.70, 150.60, 142.05,
159.82, 149.74, 135.03, 133.93, 132.24, 131.46, 131.07, 130.16, 129.28,
126.62, 126.09, 125.33, 123.89, 92.52, 26.55, 20.93, 18.35; HRMS:
calcd. for C20H19BrN4O2: 449.0589 [MþNa]þ, found: 449.0591.
140.12, 137.94, 128.50, 127.43, 123.28, 122.77, 121.48, 119.23, 116.26,
115.09, 92.36, 64.54, 21.82, 14.90; HRMS: calcd. for C19H18BrN3O2:
422.0480 [MþNa]þ, found: 422.0480.
4.10.1.8. 2-((5-Bromo-2-((4-chloro-2-fluorophenyl)amino)pyr-
imidin-4-yl)oxy)-N-methylbenzamide (3h). White powder; Yield
4.10.1.14. 5-Bromo-4-(3-fluorophenoxy)-N-(m-tolyl)pyrimidin-2-
amine (3n). White solid; Yield 68%; m.p. 125e127 ꢀC; 1H NMR
88%; m.p. 157e159 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.27 (s, 1H),
(400 MHz, DMSO‑d6)
d
9.73 (s, 1H), 8.54 (s, 1H), 7.54 (dd, J ¼ 15.1,
8.48 (s, 1H), 8.12 (d, J ¼ 4.6 Hz, 1H), 7.60 (dd, J ¼ 7.6, 1.3 Hz, 1H), 7.55
(td, J ¼ 7.9, 1.5 Hz, 1H), 7.45 (t, J ¼ 8.8 Hz, 1H), 7.39 (d, J ¼ 7.5 Hz, 1H),
7.37e7.31 (m, 2H), 6.94 (d, J ¼ 8.1 Hz, 1H), 2.64 (d, J ¼ 4.6 Hz, 3H);
8.2 Hz, 1H), 7.33 (dt, J ¼ 9.9, 2.2 Hz, 1H), 7.23 (s, 1H), 7.21 (dd, J ¼ 8.8,
2.1 Hz, 1H), 7.17 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.12 (d, J ¼ 7.7 Hz, 1H), 6.96
(t, J ¼ 7.8 Hz, 1H), 6.69 (d, J ¼ 7.4 Hz, 1H), 2.08 (s, 3H); 13C NMR
13C NMR (100 MHz, DMSO‑d6)
d
166.03, 165.28, 160.70, 158.59,
(100 MHz, DMSO‑d6)
d
164.95, 162.96 (d, J ¼ 245.0 Hz), 161.09,
155.77, 149.71, 131.60, 130.12, 129.39, 126.65, 126.54, 126.39, 125.81,
124.27, 124.05, 116.37, 94.12, 26.48; HRMS: calcd. for
158.62, 153.74, 139.96, 137.94, 131.49, 128.50, 123.04, 119.54, 118.79,
116.62, 113.38, 110.69, 92.64, 21.67; HRMS: calcd. for C17H13BrFN3O:
374.0304 [MþH]þ, found: 374.0306.
C
18H13BrClFN4O2: 472.9792 [MþNa]þ, found: 472.9790.

D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
13
4.10.1.15. 5-Bromo-4-(3,5-dichlorophenoxy)-N-(m-tolyl)pyrimidin-
DMSO‑d6) d 163.96, 161.86, 158.22, 153.71, 146.30, 145.20, 139.17,
2-amine (3o). White solid; Yield 82%; m.p. 163e165 ꢀC; 1H NMR
131.50 (2C), 125.95, 121.56, 121.32 (2C), 114.17, 113.67, 92.58; HRMS:
(400 MHz, DMSO‑d6)
d
9.78 (s, 1H), 8.55 (s, 1H), 7.64 (s, 1H), 7.57 (s,
calcd. for C16H9Br2FN4O3: 482.9104 [MþH]þ, found: 482.9103.
2H), 7.20 (s, 1H), 7.15 (d, J ¼ 7.5 Hz, 1H), 7.00 (t, J ¼ 7.7 Hz, 1H), 6.72
(d, J ¼ 7.3 Hz, 1H), 2.11 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
4.10.1.22. 4-(2-Fluoro-4-nitrophenoxy)-N-(3,4,5-trimethoxyphenyl)
d
164.79, 161.25, 158.53, 153.93, 139.92, 137.96, 135.06 (2C), 128.53,
pyrimidin-2-amine (5a). White solid; Yield 58%; m.p. 156e158 ꢀC;
126.45, 123.16, 122.32 (2C), 119.60, 116.65, 92.54, 21.59; HRMS:
1H NMR (400 MHz, DMSO‑d6)
d 9.67 (s, 1H), 8.63 (s, 1H), 8.41 (dd,
calcd. for C17H12BrCl2N3O: 423.9619 [MþH]þ, found: 423.9614.
J ¼ 10.1, 2.6 Hz, 1H), 8.23 (dd, J ¼ 9.0, 2.7 Hz, 1H), 7.91e7.78 (m, 1H),
6.79 (s, 2H), 3.57 (s, 3H), 3.56 (s, 3H), 3.32 (s, 3H). 13C NMR
4.10.1.16. 5-Bromo-4-(3-nitrophenoxy)-N-(m-tolyl)pyrimidin-2-
(100 MHz, DMSO‑d6) d 163.73, 161.73, 158.58, 154.77, 153.04 (2C),
amine (3p). White solid; Yield 77%; m.p. 164e166 ꢀC; 1H NMR
152.27, 145.93, 145.22, 135.65, 133.97, 125.57, 121.48, 113.74, 98.67,
92.20, 60.56, 56.37 (2C). HRMS: calcd. for C19H17FN4O6: 417.1205
[MþH]þ, found: 417.1205.
(400 MHz, DMSO‑d6)
d 9.74 (s, 1H), 8.57 (s, 1H), 8.31e8.18 (m, 2H),
7.89e7.76 (m, 2H), 7.14 (s, 1H), 7.08 (d, J ¼ 7.0 Hz, 1H), 6.91 (t,
J ¼ 7.7 Hz, 1H), 6.67 (d, J ¼ 7.3 Hz, 1H), 2.02 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6)
d
164.83, 161.28, 158.50, 153.07, 149.01, 139.83,
4 .10 .1. 2 3 . N 4 - ( 3 - a m i n o p r o p y l ) - 5 - b r o m o - N 2 - ( 3 , 4 , 5 -
137.86, 131.62, 129.75, 128.43, 123.15, 121.35, 119.63, 118.26, 116.70,
92.68, 21.58; HRMS: calcd. for C17H13BrN4O3: 401.0249 [MþH]þ,
found: 401.0248.
trimethoxyphenyl)pyrimidine-2,4-diamine (8a). Yellow powder;
Yield 70%; m.p. 123e125 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d8.14 (s,
1H), 7.68 (s, 1H), 7.10 (s, 2H), 6.98 (s, 1H), 3.77 (s, 6H), 3.63 (s, 3H),
3.27e3.23 (m, 2H), 2.67 (t, J ¼ 6.0 Hz, 2H), 1.55e1.40 (m, 2H), 0.99
4.10.1.17. 5-Bromo-4-(4-bromophenoxy)-N-(m-tolyl)pyrimidin-2-
(d, J ¼ 6.1 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6)
d 163.87, 161.53,
amine (3q). Yellow solid; Yield 91%; m.p. 186e188 ꢀC; 1H NMR
158.49, 152.86 (2C), 135.81, 135.79, 119.28, 116.29, 100.05, 60.56,
56.31 (2C), 27.43, 25.95 (2C). HRMS: calcd. for C16H22BrN5O3:
412.0979 [MþH]þ, found: 412.0977.
(400 MHz, DMSO‑d6)
d
9.72 (s, 1H), 8.53 (s, 1H), 7.69 (d, J ¼ 8.8 Hz,
2H), 7.29 (d, J ¼ 8.8 Hz, 2H), 7.22 (s, 1H), 7.07 (d, J ¼ 6.9 Hz, 1H), 6.97
(t, J ¼ 7.8 Hz, 1H), 6.69 (d, J ¼ 7.3 Hz, 1H), 2.08 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6)
d
165.06,161.05,158.56,152.06,139.96,137.99,
4.10.1.24. N4-(3-aminopropyl)-N2-(3,4,5-trimethoxyphenyl)pyrimi-
133.16 (2C), 128.48, 125.01 (2C), 123.01, 119.43,118.86, 116.59, 92.59,
21.74; HRMS: calcd. for C17H13Br2N3O: 433.9504 [MþH]þ, found:
433.9502.
dine-2,4-diamine (8b). White solid; Yield 68%; m.p. 120e122 ꢀC; 1H
NMR (400 MHz, DMSO‑d6)
d
8.89 (s, 1H), 7.89 (d, J ¼ 7.1 Hz,1H), 7.83
(s, 1H), 7.12 (s, 2H), 6.30 (d, J ¼ 7.1 Hz, 1H), 3.80 (s, 6H), 3.66 (s, 3H),
2.82 (s, 2H), 1.68e1.54 (m, 4H). 13C NMR (100 MHz, DMSO‑d6)
4.10.1.18. 4-(4-(benzyloxy)phenoxy)-5-bromo-N-(m-tolyl)pyrimidin-
d 161.10, 159.51, 154.60, 153.26 (2), 134.90, 134.38, 118.98, 116.02,
2-amine (3r). White solid; Yield 65%; m.p. 178e180 ꢀC; 1H NMR
99.38, 60.60, 56.32 (2), 25.71, 24.90 (2) HRMS: calcd. for
(400 MHz, DMSO‑d6)
d
9.67 (s, 1H), 8.50 (s, 1H), 7.49 (d, J ¼ 7.1 Hz,
C
16H23N5O3: 334.1874 [MþH]þ, found: 334.1873.
2H), 7.43 (t, J ¼ 7.2 Hz, 2H), 7.40e7.32 (m, 1H), 7.29e7.17 (m, 3H),
7.12 (d, J ¼ 8.7 Hz, 3H), 6.91 (t, J ¼ 7.6 Hz, 1H), 6.67 (d, J ¼ 7.0 Hz, 1H),
4.10.1.25. N4-(3-aminopropyl)-5-iodo-N2-(3,4,5-trimethoxyphenyl)
5.13 (s, 2H), 2.07 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
165.51,
pyrimidine-2,4-diamine (8c). Yellow powder; Yield 65%; m.p.
160.74, 158.66, 156.61, 146.19, 140.06, 137.97, 137.45, 128.97 (2C),
128.50, 128.41, 128.24 (2C), 123.42 (2C), 122.87, 119.35, 116.49,
116.03 (2C), 92.66, 70.09, 21.74; HRMS: calcd. for C24H20BrN3O2:
462.0817 [MþH]þ, found: 462.0815.
111e113 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d8.30 (s, 1H), 7.78 (s, 2H),
7.01 (s, 2H), 3.78 (s, 6H), 3.66 (s, 3H), 3.31 (d, J ¼ 5.2 Hz, 2H),
2.84e2.69 (m, 2H), 1.77e1.73 (m, 2H). 1H NMR (100 MHz, DMSO‑d6)
d
159.63, 159.30, 158.96, 158.64, 152.90 (2C), 118.60, 115.65, 102.01,
96.40, 60.60, 56.46 (2C), 38.44, 37.13 (2C). HRMS: calcd. for
4.10.1.19. 5-Bromo-4-(2-fluoro-4-nitrophenoxy)-N-(3,4,5-
trimethoxyphenyl)pyrimidin-2-amine (3s). Yellow powder; Yield
C
16H22IN5O3: 460.0840 [MþH]þ, found: 460.0842.
87%; m.p. 190e193 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.68 (s, 1H),
4.10.1.26. N4-(2-(diethylamino)ethyl)-N2-(3,4,5-trimethoxyphenyl)
8.63 (s, 1H), 8.42 (d, J ¼ 8.6 Hz, 1H), 8.23 (d, J ¼ 8.5 Hz, 1H), 7.84 (t,
pyrimidine-2,4-diamine (10a). White solid; Yield 77%; m.p.
J ¼ 8.3 Hz, 1H), 6.78 (s, 2H), 3.56 (s, 6H), 3.55 (s, 3H); 13C NMR
114e106 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.09 (s, 1H), 7.81 (d,
(100 MHz, DMSO‑d6)
d
163.74, 161.74, 158.59, 153.53, 153.06 (2C),
J ¼ 5.6 Hz, 1H), 7.10 (s, 2H), 6.36 (s, 1H), 5.97 (d, J ¼ 5.7 Hz, 1H), 3.78
(s, 6H), 3.61 (s, 3H), 3.37e3.33 (m, 2H), 2.56e2.51 (m, 2H),
2.49e2.43 (m, 4H), 0.95e0.90 (m, 6H). 13C NMR (100 MHz,
145.98, 145.30, 135.68, 133.93, 125.59, 121.51, 113.65, 98.62 (2C),
92.22, 60.57, 56.36 (2C); HRMS: calcd. for C19H16BrFN4O6: 517.0135
[MþNa]þ, found: 517.0135.
DMSO‑d6)
d 162.35, 161.49, 161.03, 156.50, 153.12 (2), 137.17, 132.67,
97.54 (2), 60.55, 56.15 (2), 52.30, 52.01, 47.08 (2), 12.35 (2). HRMS:
4.10.1.20. 5-Bromo-N-(4-chlorophenyl)-4-(2-fluoro-4-nitrophenoxy)
pyrimidin-2-amine (3t). Yellow powder; Yield 70%; m.p.
calcd. for C19H29N5O3: 376.2343 [MþH]þ, found: 376.2341.
175e178 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
10.01 (s, 1H), 8.66 (s,
4.10.1.27. N4 -(2-(diethylamino)ethyl)-5-iodo-N2-(3,4,5-
1H), 8.48 (d, J ¼ 8.6 Hz, 1H), 8.27 (d, J ¼ 8.3 Hz, 1H), 7.84 (t,
trimethoxyphenyl)pyrimidine-2,4-diamine (10b). Yellow powder;
J ¼ 8.2 Hz, 1H), 7.35 (s, 2H), 7.14 (d, J ¼ 7.5 Hz, 2H); 13C NMR
Yield 72%; m.p. 101e103 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
8.13 (s,
1H), 7.69 (s, 1H), 7.05 (s, 2H), 6.66 (s, 1H), 3.77 (s, 6H), 3.62 (s, 3H),
3.29e3.24 (m, 2H), 2.46e2.48 (m, 2H), 2.45 (s, 4H), 0.88 (s, 6H). 13
NMR (100 MHz, DMSO‑d6) 163.92, 158.57, 156.01, 155.59, 152.84
(100 MHz, DMSO‑d6) d 163.96, 161.87, 158.26, 153.71, 146.30, 145.20,
138.75, 128.61 (2C), 126.22, 125.95, 121.57, 120.93 (2C), 113.67,
92.53; HRMS: calcd. for C16H9BClFN4O3: 438.9609 [MþH]þ, found:
438.9606.
C
d
(2C), 135.67, 133.85, 100.19 (2C), 60.50, 56.27 (2C), 52.04, 50.55,
47.07 (2C), 12.28 (2C). HRMS: calcd. for C19H28IN5O3: 502.1310
[MþH]þ, found: 502.1313.
4.10.1.21. 5-Bromo-N-(4-bromophenyl)-4-(2-fluoro-4-nitrophenoxy)
pyrimidin-2-amine (3u). Yellow powder; Yield 76%; m.p.
163e165 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
10.01 (s, 1H), 8.66 (s,
Declaration of competing interest
1H), 8.48 (dd, J ¼ 10.1, 2.5 Hz, 1H), 8.26 (dd, J ¼ 8.9, 1.4 Hz, 1H),
7.94e7.75 (m, 1H), 7.27 (t, J ¼ 9.7 Hz, 4H); 13C NMR (100 MHz,
The authors declare that they have no known competing

14
D. Sun et al. / European Journal of Medicinal Chemistry 208 (2020) 112782
verteporfin, J. Biol. Chem. 286 (2011) 7290e7300.
[14] S. Yang, X. Wang, G. Contino, Pancreatic cancers require autophagy for tumor
growth, Genes Dev. 25 (2011) 717e729.
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[15] C.M. Dower, N. Bhat, M.T. Gebru, L. Chen, C.A. Wills, B.A. Miller, H.G. Wang,
Targeted inhibition of ULK1 promotes apoptosis and suppresses tumor
growth and metastasis in neuroblastoma, Mol. Canc. Therapeut. 17 (2018)
2365e2376.
Acknowledgments
We are grateful to Prof. Liang Ouyang and Prof. Jie Liu (Sichuan
University) for their critical reviews on this work. This work was
financially supported by National Natural Science Foundation of
China (Grant No. 81970481), Sichuan Science and Technology
Program (Grant No. 2020JDRC0053), Liaoning Province Natural
Science Foundation (Grant No. 2019-MS-299) and Liaoning Revi-
talization Talents Program (Grant No. XLYC1807182).
[16] E.Y. Chan, A. Longatti, N.C. McKnight, S.A. Tooze, Kinase-inactivated ULK
proteins inhibit autophagy via their conserved C-terminal domains using an
Atg13-independent mechanism, Mol. Cell Biol. 29 (2009) 157e171.
[17] F. Tang, P.C. Hu, Z.T. Yang, C. Xue, J. Gong, S.X. Sun, L. Shi, S.M. Zhang, Z.Z. Li,
C.X. Yang, J.H. Zhang, C.N. Xie, SBI0206965, a novel inhibitor of Ulk1, sup-
presses non-small cell lung cancer cell growth by modulating both autophagy
and apoptosis pathways, Oncol. Rep. 37 (2017) 3449e3458.
[18] L. Zhang, L. Ouyang, Y. Guo, J. Zhang, B. Liu, UNC-51-like kinase 1: from an
autophagic initiator to multifunctional drug target, J. Med. Chem. 61 (15)
(2018) 6491e6500.
[19] M.B. Lazarus, C.J. Novotny, K.M. Shokat, Structure of the human autophagy
initiating kinase ULK1 in complex with potent inhibitors, ACS Chem. Biol. 10
(2015) 257e261.
[20] M.B. Lazarus, K.M. Shokat, Discovery and structure of a new inhibitor scaffold
of the autophagy initiating kinase ULK1, Bioorg. Med. Chem. 23 (2015)
5483e5488.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112782.
[21] K.J. Petherick, O.J. Conway, C. Mpamhanga, S.A. Osborne, A. Kamal, B. Saxty,
I.G. Ganley, Pharmacological inhibition of ULK1 kinase blocks mammalian
target of rapamycin (mTOR)-dependent autophagy, J. Biol. Chem. 290 (2015)
11376e11383.
[22] S. Drießen, N. Berleth, O. Friesen, A.S. Loffler, P. Bohler, N. Hieke, B. Stork,
Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade
of autophagy, Autophagy 11 (2015) 1458e1470.
[23] D. F Egan, M.G. Chun, M. Vamos, H.X. Zou, J. Rong, C.J. Miller, R.J. Shaw, Small
molecule inhibition of the autophagy kinase ULK1 and identification of ULK1
substrates, Mol. Cell. 59 (2015) 285e297.
References
[1] K.D. Miller, L. Nogueira, A.B. Mariotto, J.H. Rowland, K.R. Yabroff, C.M. Alfano,
A. Jemal, J.L. Kramer, R.L. Siegel, Cancer treatment and survivorship statistics,
CA A Cancer J. Clin. 5 (2019) 363e385.
[2] W.D. Travis, E. Brambilla, A.P. Burke, A. Marx, A.G. Nicholson, WHO classifi-
cation of tumours of the lung, pleura, thymus and heart. Lyon: international
agency for research on cancer, J. Thorac. Oncol. 10 (2015) 1383e1395.
[3] K. Politi, R.S. Herbst, Lung cancer in the era of precision medicine, Clin. Canc.
Res. 21 (2015) 2213e2220.
[4] N. Duma, R. Santana-Davila, J.R. Molina, Non-small cell lung cancer: epide-
miology, screening, diagnosis, and treatment, Mayo Clin. Proc. 94 (8) (2019)
1623e1640.
[5] L. Osmani, F. Askin, E. Gabrielson, Q.K. Li, Current WHO guidelines and the
critical role of immunohistochemical markers in the subclassification of non-
small cell lung carcinoma (NSCLC): moving from targeted therapy to immu-
notherapy, Semin. Canc. Biol. 52 (2018) 103e109.
[6] B. Levine, D.J. Klionsky, Development by self-digestion: molecular mecha-
nisms and biological functions of autophagy, Dev. Cell 6 (2004) 463e477.
[7] B. Ravikumar, R. Duden, D.C. Rubinsztein, Aggregateprone proteins with
polyglutamine and polyalanine expansions are degraded by autophagy, Hum.
Mol. Genet. 11 (2002) 1107e1117.
[8] Y. Kabeya, Y. Kamada, M. Baba, H. Takikawa, M. Sasaki, Y. Ohsumi, Atg17
functions in cooperation with Atg1 and Atg13 in yeast autophagy, Mol. Biol.
Cell 16 (2005) 2544e2553.
[9] J.M. Park, M. Seo, C.H. Jung, D. Grunwald, M. Stone, N.M. Otto, E. Toso, Y. Ahn,
M. Kyba, T.J. Griffin, L. Higgins, D.H. Kim, ULK1 phosphorylates Ser30 of BECN1
in association with ATG14 to stimulate autophagy induction, Autophagy 14
(2018) 584e597.
[10] O. Kapuy, P.K. Vinod, G. Banhegyi, mTOR inhibition increases cell viability via
autophagy induction during endoplasmic reticulum stress - an experimental
and modeling study, FEBS Open Bio 4 (2014) 704e713.
€
€
[24] S. Porey, X. Zhang, S. Bhowmick, V. Kumar Singh, S. Guin, R.S. Paton, D. Maiti,
An alkyne linchpin strategy for drug: pharmacophore conjugation: experi-
mental and computational realization of a meta-selective inverse sonogashira
coupling, J. Am. Chem. Soc. (2020), https://doi.org/10.1021/jacs.9b10646.
[25] C. Chang, S. Ekins, P. Bahadduri, P.W. Swaan, Pharmacophore-based discovery
of ligands for drug transporters, Adv. Drug Deliv. Rev. 58 (2006) 1431e1450.
[26] D.J. Sun, Y.Q. Zhao, S.Y. Zhang, L. Zhang, B. Liu, L. Ouyang, Dual-target kinase
drug design: current strategies and future directions in cancer therapy, Eur. J.
Med. Chem. (2020), https://doi.org/10.1016/j.ejmech.2019.112025.
ꢀ
[27] J.P. Metivier, B. Cuissart, R. Bureau, A. Lepailleur. The Pharmacophore
Network:
A Computational Method for Exploring Structure-Activity Re-
lationships from a Large Chemical Data Set.
[28] B.M. Lazarus, C.J. Novotny, K.M. Shokat, Structure of the human autophagy
initiating kinase ULK1 in complex with potent inhibitors, ACS Chem. Biol. 10
(2015) 257e261.
[29] D.B. Kitchen, Docking and scoring in virtual screening for drug discovery:
methods and applications, Nat. Rev. Drug Discov. 3 (2004) 935e949.
[30] G. Wu, D.H. Robertson, C.L. Brooks, M. Vieth, J. Comput. Chem. 24 (2003)
1549e1562.
[31] L. Ouyang, L. Zhang, S. Zhang, D. Yao, Y. Zhao, G. Wang, L. Fu, P. Lei, B. Liu,
Small-molecule activator of UNC-51-Like kinase 1 (ULK1) that induces cyto-
protective autophagy for Parkinson’s disease treatment, J. Med. Chem. 61
(2018).
[32] L. Ouyang, L. Zhang, L. Fu, B. Liu, A small-molecule activator induces ULK1-
modulating autophagy-associated cell death in triple negative breast cancer,
Autophagy 13 (2017) 777e778.
[33] D. Sun, L. Zhu, Y. Zhao, Fluoxetine induces autophagic cell death via eEF2K-
AMPKmTOR-ULK complex axis in triple negative breast cancer, J. Cell Prolif-
eration. 51 (2017) 12402e12412.
[11] S. Shoji-Kawata, R. Sumpter, M. Leveno, et al., Identification of a candidate
therapeutic autophagy inducing peptide, Nature 494 (2013) 201e206.
[12] T.T. Shi, X.X. Yu, L.J. Yan, H.T. Xiao, Research progress of hydroxychloroquine and
autophagy inhibitors on cancer, Canc. Chemother. Pharmacol. 79 (2017) 287e294.
[13] E. Donohue, A. Tovey, A.W. Vogl, S. Arns, E. Sternberg, R.N. Young, M. Roberge,
Inhibition of autophagosome formation by the benzoporphyrin derivative