Discovery of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine derivatives that regulated HPV relevant cellular pathway and prevented cervical cancer from abnormal proliferation

Article abstract of DOI:10.1016/j.ejmech.2020.112556

Human papillomavirus (HPV) is a well-established etiological factor for cervical cancer, and the expression of oncogenic protein E7 is crucial for carcinogenesis. Herein, virtual screening was performed and 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine derivatives were designed, synthesized as antineoplastic agents, and evaluated for their anti-tumor activities. Among them, the most promising compound H1 showed specific anti-proliferation ability against HeLa cells (IC₅₀ = 380 nM) as well as excellent inhibition of tumor growth in the HeLa xenograft model without inducing obvious side effects. It is interesting that compound H1 displayed significant inhibition against HPV18-positive cervical cell lines (HeLa) but not for HPV16-positive cervical cell lines (SiHa). Further study demonstrated that a low concentration of compound H1 could lead to a cell cycle blockage at the G1 phase and promote cell apoptosis slightly (8.77%). Compound H1 also exhibited transcription repression, especially those associated with the oncoprotein E7 cellular pathway like E7/Rb/E2F-1/DNMT1, which were essential in tumorigenesis. Proteomics analysis revealed that E7 might be degraded through E3 ubiquitin ligases, which aligned with decreasing expression of E7 following the treatment of compound H1. Taken together, it indicated that compound H1 could be a promising potential agent for cervical cancer treatment.

Full text of DOI:10.1016/j.ejmech.2020.112556

Journal Pre-proof
Discovery of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine derivatives
that regulated HPV relevant cellular pathway and prevented cervical cancer from
abnormal proliferation
Peili Jiao, Yuxi Wang, Beibei Mao, Bingding Wang, Yi Zhong, Hongwei Jin, Lihe
Zhang, Liangren Zhang, Zhenming Liu
PII:
S0223-5234(20)30528-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112556
EJMECH 112556
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2020
Revised Date: 19 May 2020
Accepted Date: 7 June 2020
Please cite this article as: P. Jiao, Y. Wang, B. Mao, B. Wang, Y. Zhong, H. Jin, L. Zhang, L. Zhang, Z.
Liu, Discovery of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine Derivatives that Regulated
HPV Relevant Cellular Pathway and Prevented Cervical Cancer from Abnormal Proliferation, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112556.
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Graphic Abstract

Discovery of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine
Derivatives that Regulated HPV Relevant Cellular Pathway and
Prevented Cervical Cancer from Abnormal Proliferation
a
a
b
a
a
a
†
†
Peili Jiao , , Yuxi Wang , , Beibei Mao , Bingding Wang , Yi Zhong , Hongwei Jin ,
a
a
a
Lihe Zhang , Liangren Zhang ,*, Zhenming Liu ,*
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing, 100191, China;
b
College of Pharmaceutical Science, Shandong University of Traditional Chinese
Medicine, Jinan, Shandong, China.
Author Email Address:
Peili Jiao: pelly@bjmu.edu.cn;
Yuxi Wang: yuxiwang@bjmu.edu.cn;
Beibei Mao: maobeibei89@163.com;
Hongwei Jin: jinhw@bjmu.edu.cn;
Bingding Wang: 1710307424@pku.edu.cn;
Yi Zhong: yi_zhong@pku.edu.cn
Lihe Zhang: zdszlh@bjmu.edu.cn;
Liangren Zhang: liangren@bjmu.edu.cn;
Zhenming Liu: zmliu@bjmu.edu.cn.
†
:
:
These authors contributed equally.
To whom correspondence should be addressed.
*
Liangren, Zhang, Ph. D
Professor
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University
Beijing 100191, China
Tel: +86-10-82802567
Fax: +86-10-82802724
Email: liangren@bjmu.edu.cn
Zhenming Liu, Ph. D
Associate Professor
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University
Beijing 100191, China
Tel: +86-10-82805514

Fax: +86-10-82802724
Email: zmliu@bjmu.edu.cn

Abstract
Human papillomavirus (HPV) is a well-established etiological factor for cervical
cancer, and the expression of oncogenic protein E7 is crucial for carcinogenesis.
Herein, virtual screening was performed and 2-(2-aminobenzo[d]thiazol-6-yl)
benzo[d]oxazol-5-amine derivatives were designed, synthesized as antineoplastic
agents, and evaluated for their anti-tumor activities. Among them, the most promising
compound H1 showed specific anti-proliferation ability against HeLa cells (IC =
5
0
3
80 nM) as well as excellent inhibition of tumor growth in the HeLa xenograft model
without inducing obvious side effects. It is interesting that compound H1 displayed
significant inhibition against HPV18-positive cervical cell lines (HeLa) but not for
HPV16-positive cell lines (SiHa). Further study demonstrated that a low
concentration of compound H1 could lead to a cell cycle blockage at the G1 phase
and promote cell apoptosis slightly (8.77%). Compound H1 also exhibited
transcription repression, especially those associated with the oncoprotein E7 cellular
pathway like E7/Rb/E2F-1/DNMTI, which were essential in tumorigenesis.
Proteomics analysis revealed that E7 might be degraded through E3 ubiquitin ligases,
which aligned with decreasing expression of E7 following the treatment of compound
H1. Taken together, it indicated that compound H1 could be a promising potential
agent for cervical cancer treatment.
Keywords: Cervical cancer, Human papillomavirus E7,
2
-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine derivatives, Cellular
pathway
Highlights:
ó Identification of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine
derivatives and compound H1 was identified as a specific inhibitor for
HPV18-positive cervical cancer cells.
ó The compound H1 inhibited expression of HPV E7 protein which associated with
cell proliferation process, preventing cervical cancer cells from premature cell
procession and abnormal proliferation.
ó E7-Rb-E2F cellular pathway and DNA methylation were suppressed by compound
H1.
ó Proteomics analysis revealed that E7 might be degraded through E3 ubiquitin
ligases, which aligned with decreasing expression of E7 following the treatment of
compound H1.

Graphic Abstract

Introduction
Among females, cervical cancer ranks as the fourth most frequently diagnosed cancer
and the fourth leading cause of cancer death for both incidence and mortality [1].
Despite the treatment of surgery, radiotherapy, chemotherapy, and vaccines for
cervical cancer have made a great improvement, the prognosis of patients with
malignant or metastatic cervical cancer remains poor [2]. It has been established that
high-risk types of human papillomavirus (HPV) are the main etiological factors for
cervical cancer [3, 4]. HPV integrates DNA into the cellular genome and encodes
oncoproteins [5] which manipulates gene expression patterns in the host cell to
facilitate viral replication [6], regulating viral transcription, and promoting immune
evasion and persistence [7-9]. Previous research work shows that the expression of
oncoprotein E7 is crucial for both the initiation and the maintenance of cervical
cancer.
Viral oncogenicity is largely related to their encoded E7 proteins, which could
influence and inhibit the host tumor suppressor retinoblastoma protein (Rb) [10-12].
The E2 factor (E2F) family of transcription factors are downstream targets of
hyperphosphorylated Rb and it has been known for several years to be important
regulators of S-phase entry [13]. E7 binds to the complex of Rb and E2F will cause
the release of E2F, resulting in the dissociation of Rb from E2F transcription factors,
following by the arrestment of cell cycle in the G phase [7] and abrogation of cell
1
cycle checkpoints. In cells persistently infected with HPV, this lead to the
accumulation of cellular mutations over time and the consequent progression to
cancer [14].
Besides, it is discovered that epigenetic modifications also participate in the cancer
development, leading to uncontrolled cell proliferation [15]. One of the most widely
studied epigenetic mechanisms is DNA methylation, a reversible reaction catalyzed
by DNA methyltransferase (DNMT) [16]. DNMT1 is
methyltransferase that preserves the methylation pattern during each cellular division
17-19]. The E7 oncoproteins increases the expression and activity of DNMTs,
a
maintenance
[
particularly DNMT1 [7]. The binding of E7 to DNMT1 (E7/DNMT1) induces a
conformational change of DNMT1, exposing its DNA binding site and promoting
DNA binding; and then DNMT1 closes on the DNA and maintains a stable
DNMT1/DNA interaction. Meanwhile, E7 dissociates from the complex [20-23] and
enters the next loop. The release of E2F mentioned above also positively regulates the
promoter activity of DNMT1 [20]. Overexpression of DNMT1 results in
hypermethylation of tumor-suppressor gene promoters, which lead to cellular
transformation and tumorigenesis [20, 23].
Herein, we report a series of structures obtained via substructure virtual searching and
the studies of structure-activity relationships (SARs) driven by multi-stage activity
evaluation to discover novel antineoplastic agents on cervical cancer. The anticancer
activities and safety of the promising lead compound H1 were characterized, and

mechanistic studies were also further investigated. Compound H1 showed a specific
anti-proliferation ability in the treatment of HPV18-positive cervical cancer, which
was the unique phenotype in reported anticancer compounds. Further research on cell
cycle and apoptosis revealed that compound H1 could repress the cell proliferation
process and cellular signaling pathways associated with tumorigenesis.

Results and discussion
Compound H1 inhibited the proliferation of HPV18-positive cervical cancer
In the previous study, we accidentally found compound H1 presented remarkable
cervical cancer inhibition. To verify the selectivity of compound H1 towards cervical
cancer, proliferative inhibition test was conducted on different kinds of cancer cell
lines, including HeLa (HPV18-positive cervical cancer cell), SiHa (HPV16-positive
cancer cell), SKOV-3 (human ovarian cancer cell), MCF7 (human breast cancer cell),
A375 (human melanoma cell), A549 (human lung adenocarcinoma cell), U87 (human
glioblastoma cell), NCI-H460 (human large-cell large carcinoma) and A431 (human
epidermoid carcinoma cell). The results showed that compound H1 had significant
inhibition toward HeLa cells (IC = 380 nM) over other cancer cell lines and it also
5
0
showed low toxicity in HEK-293 (human embryonic kidney cells 293) cells (IC >
5
0
2
0 μM) (Fig. 1A). Surprisingly, as shown in Fig. 1B, compound H1 displayed
significant inhibition against HPV18-positive cervical cells of HeLa (IC = 380 nM)
5
0
and C4-I (IC = 440 nM). In contrast, HPV16-positive cervical cancer cells (SiHa)
5
0
and HPV-negative cervical cancer cells (C-33A) were less sensitive toward the
treatment of compound H1 with less proliferative repression.
Fig.1| Compound H1 displayed a significant anti-cervical cell proliferation in vitro.
(A) Anti-proliferative activities of compound H1 against various cancer cell lines. (B)
The effect on cell viability of four types of cervical cancer cell lines of compound H1.
Data were presented as mean ± SE (n = 3).
The structure-activity relationship of compound H1
To investigate the structure-activity relationships (SARs) of compound H1, a
substructure-based virtual searching was performed to screen 300,000 compounds
from SPECS chemical library and Chinese National Compound Library of Peking
University (PKU-CNCL). The substructure queries contained benzoxazole,
benzothiazole, benzimidazole and benzotriazole. Eventually, 64 compounds were

selected manually from hits and their proliferative inhibition was tested via
Sulforhodamine B (SRB) assay in HeLa cells.
Tab. 1 The Hela cells proliferative inhibition rate of benzoxazole, benzothiazole,
benzimidazole and benzotriazole derivatives
Compound
R1
R2
Inh. % (20 μM)
1
，H1
5-NH
2
81.1
2
3
4
5
6
H
19.3
-0.7
-2.3
3.4
4-NH
4-NH
4-NH
5-NH
2
2
2
2
3.7
7
5-NH
2
42.4
8
9
5-NH
5-NH
5-NH
2
2
2
42.6
1.2
1
0
8.9
1
1
5-NH
2
2
5.8
6.0
1
2
5-NH

1
3
5-NH
2
-0.2
1
1
1
4
5
6
5-NH
5-NH
5-NH
2
2
2
3.2
26.5
1.3
1
1
7
8
5-NH
2
2
2
13.8
3.8
5-NO
19
20
21
22
23
24
25
26
27
28
29
30
31
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
2
2
2
2
2
2
2
2
2
2
3-Cl
5.3
6.9
-0.3
-3.4
6.0
9.1
9.6
12.2
0.6
1.7
6.1
2.5
7.9
4-NH
4-CH
4-OCH
4-NO
4-SOCH
5-NH
3
3
2
3
2
5-OCH
6-OCH
6-Br
3
3
CH
SO
SH
5-NH
3
2
H
3
H
3
2
H
2.6

3
3
H
4.9
3
3
3
3
3
3
4
4
5
6
7
8
9
0
H
H
H
H
H
H
H
8.1
1.7
2.6
4.4
5.5
0.1
10.8
4
4
1
2
H
H
0.3
0.8
4
4
3
4
H
H
3.0
4.4

4
5
H
47.8
46
47
48
49
H
H
H
H
1.2
2.1
9.7
6.6
5
5
0
1
H
H
3.5
5.5
5
2
3
NH
2
H
H
1.1
1.9
5
5
4
5
H
H
0.0
0.2
5
5
6
H
34.5

57
58
59
H
2.5
4.0
5-NH
5-NH
2
53.0
2
6
0
5-NH
2
7.1
#
Inh. % represents the inhibition rate in the SRB assay. Results are showed as a
mean of at least two replicates.
As shown in Tab. 1, the proliferative inhibition of compounds obtained via virtual
screening was not strong enough generally, and only 4 of which (7, 8, 45, 59) had
more than 40% inhibition rate at 20 μM in the SRB assay. Notwithstanding, some
SAR rules still could be concluded from the virtual screening results. The
benzothiazole (H1, 45) was indispensable since the inhibition activity declined
significantly when the R was replaced by NH (19 - 28), alkyl (29), SO (30), SH (31),
1
2
3
substituted phenyl or pyridyl (16, 44, 46, 47) group. For the R moiety, the 5-NH
2
2
group (H1, 7, 8) was beneficial for the inhibition activity, but the activity disappeared
when the R position was substituted by the 4-NH group.
2
2
Synthesis of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d] oxazole-5-amine
derivatives
Several compounds were synthesized to investigate the beneficial location of NH in
2
the R or R . The synthesis of compound H1 and compound 61 - 64 can be divided
1
2
into two steps (Fig.2). Firstly, benzo[d]thiazole-6-carboxylic acid derivatives and
-aminophenol derivatives were cyclized to oxazole (H1, 61-63) under acidic and
2
high-temperature conditions. Subsequently, with the triethylamine catalytic reaction,
H1, ethylamine, and N,N’-carbonyldiimidazole (CDI) were condensed to form the
1
urea group (64). According to the H-NMR of H1, it is easy to recognize the peaks of
the NH on the thiazole (δ7.86) and the benzene (δ5.07). The peaks of compound
2
6
1-63 can be assigned as well. After the condensation reaction, the peak of two
hydrogen atoms at δ5.07 disappeared, so it could be determined that the urea group
was synthesized at NH on the benzene.
2

Fig.2| The synthesis process of H1 and compound 61-64. a. Eaton's reagent, 180 ℃ ,
h; b. CDI, Et N, DMF, r.t, overnight.
8
3
As shown in Tab. 2, the IC was obtained via SRB assay (Fig. S1) in Hela cells and
5
0
it showed that the NH at the R position was indispensable, and the activity declined
2
1
remarkably when the R was replaced by a hydrogen atom (63). The NH substitution
1
2
at the R position (H1, 62) was also beneficial to the activity improvement. It should
2
be noted that the inhibition of the HeLa cells’ proliferation decreased when the R2
position was replaced by the NH . However, the R position was substituted by urea
2
2
derivatives even led to the elimination of HeLa cells inhibition. To sum up, compound
H1 was chosen for further research.
Tab.
2
Structural
optimization
of
2-(2-aminobenzo[d]thiazol-6-yl)
benzo[d]oxazol-5-amine derivatives
Inhibition Rate (%)
α
Compound R1
R2
NH
H
IC (μM)
50
2
0 μM
1 μM
1
6
6
6
(H1)
NH
2
2
81.1
49.2
67.2
58.0
81.7
0.38±0.13
6.0±5.15
1
H
17.6
59.6
20.3
2
3
NH
2
H
0.82±0.53
7.08±0.84
H
NH
2
64
NH
2
6.8
0.7
-
α
Anti-proliferative activities of selective compounds against cervical cells. Data are
b
the means ± S.E. of values from three independent experiments. “-” Not determined.

Compound H1 repressed the growth of cervical subcutaneous xenograft tumor in
nude mice
Treatment with compound H1 (50 mg/kg) on alternate days caused a significant
inhibition of HeLa tumor xenograft growth in nude mice compared with the vehicle
control (Fig.3). Unfortunately, a mouse in the HeLa group died just before the end of
the experiment. Hence, one of the tumor pictures was missing in the HeLa group, but
it did not affect the data collection. However, the other types of cancer subcutaneous
xenograft tumors showed no tumor growth inhibition after compound H1 treatment
compared with vehicle group, respectively. In addition, as reflected by unaltered body
weights, there were none of any gross signs of toxicity or possible side effects after
the treatment of compound H1. Each group was under equal experimental conditions,
including diet consumption, animal movement, administration methods and
concentrations. It is revealed that compound H1’s excellent anti-cervical cancer
ability was highly selective against other types of cancers in vivo as well.
Fig.3| Nude mice were all randomized for intragastric perfusion with 50mg/kg/ of

compound H1 every other day. Xenograft cancer models including HeLa, SKOV-3,
MCF7, A375 and A431 cell lines. Data were presented as mean ± S.E. (n = 5).
Statistical significance (p values) was calculated by a two-tailed student’s t-test. * p <
0
.05, ** p < 0.01, *** p < 0.001; test groups compared with compared with the
vehicle group.
The compound H1 modulated cell progress and repressed HPV-E7 associated
protein
To determine the effect of compound H1 on cell cycle distribution, we performed cell
cycle analysis after treating HeLa cells with compound H1 at 1 μM for 6-48 h. Flow
cytometry (FCM) analysis revealed a significant alteration on cell cycle progression.
Specifically, a conspicuous increase in the G1 phase and a concomitant decrease in
the S phase of cell cycle progression was observed after compound H1 treatment
compared to vehicle treatment at 24 h (Fig. 4A). The compound H1 also caused cell
apoptosis in cervical cancer cells, slightly increasing the apoptosis rate of HeLa cells
at a low concentration (8.77%) (Fig. 4B), as demonstrated by the FCM analysis
measuring the population of annexin V-positive cells after 24 h treatment.
To examine if H1 influenced related regulators, western blot analysis was performed.
The incubation of HeLa cells and C4-I cells (HPV18 positive cervical cancer cells)
with H1 reduced the expression level of E6 and E7 protein in a
concentration-dependent manner (Fig. 4C). Besides, the E2F/Rb complex that can
actively repress the transcription [20] is important for the regulation of genes at the
G1/S phase transition. As above mentioned, hyperphosphorylated Rb releases E2F
transcription factors and allows the expression of genes that mediate entry into the S
phase. In HeLa cells, the expression level of E7 signaling pathway proteins declined
along with HPV-E7 in response to the compound H1 treatment at indicated
concentrations for 24 hours. The expression of p-Rb (S608) (hyperphosphorylated
Rb), p-Rb (S780) (hypophosphorylated Rb), E2F-1, and DNMT1 proteins were
repressed by H1 in a dose-dependent manner (Fig. 4D). The existence of E7 results in
the phosphorylation and subsequent degradation of Rb, which dissociate the Rb/E2F
complex and cause the release of E2F, activating the transcription and driving cell
cycle progression [24]. Compound H1 treatment blocked the E7/Rb/E2F-1 cellular
signaling in cervical cancer and repressed the carcinogenic action. On the other hand,
we also observed a downward trend of E6 protein, which was involved in the
apoptosis pathway associated with p53. According to the FCM analysis, compound
H1 showed the mild ability to promote the apoptosis of cervical cancer cells, which
was aligned with western blot results. These results indicate that H1 induced a
remarkable inhibition of E7-associated proteins, and eventually led to
anti-proliferation of HeLa cells.
Moreover, the expression of DNMT1 also increases caused by the increasing level of

HPV-E7 and the release of E2F-1 protein. As mentioned above, the overexpression of
DNMT1 results in the hypermethylation of tumor-suppressor gene promoters, which
lead to cellular transformation and tumorigenesis [20]. However, the expression of
DNMT1 declined after compound H1 treatment, which indicated that compound H1
repressed E7/DNMT1/DNA cellular processes and repressed the cervical carcinogenic
process.
Fig 4| (A) H1 induced cell cycle arrest in HeLa cells in a time-dependent manner.
Cells were treated with H1 at 1μM for 6, 12, 24, 48 hours and then subjected to flow
cytometry. The bar graph shows the percentage of HeLa cells in G , S and G /M
1
2
phases. (B) Apoptosis effect of compound H1 on HeLa cells at a concentration of 1
μM. (C) Dose-dependent effect of H1 treatment on expression levels of E6, E7
protein in HPV-18 positive cells, HeLa cells and C4-I cells. (D) Expression profile of
E7 signaling pathway proteins in HeLa cells by WB analysis. Cells were treated with
the indicated concentrations of H1 for 24 h and probed for E6, E7, pRb, Rb, E2F-1
and DNMT1 proteins. Data are presented as the mean ± S.E. of three independent
experiments. Statistical significance (p values) was calculated by a two-tailed
student’s t-test. * p < 0.05, **p < 0.01, as compared with untreated cells (0 h).

Proteomics analysis
The GO (gene ontology) function significant enrichment analysis gives a GO function
entry that is enriched in the differential protein compared to all identified proteins,
thereby giving the biological function to the differential protein which is significantly
related [21]. The analysis first maps all the differential proteins to each term of the
GO database, calculating the number of proteins in each term, and then applies a
hypergeometric test to find out the GO entries significantly enriched in differential
proteins compared with all proteins.
HeLa lysates of compound H1 treatment were subjected to quantitative proteomics
analysis using label-free method. After MS analysis, a total of 5866 proteins were
identified, of which 152 proteins were found to be differential abundant (p-value <
0
.05), with 14 up-regulated proteins and 137 down-regulated ones in HeLa cell lysates
as shown in volcano plots. Selected proteins were listed as below (Fig. 5A), and the
proteasome relevant protein neuronal precursor cell-expressed developmentally
down-regulated protein 8 (NEDD8) was identified. Following the combinational
treatment with compound H1 at 5 μM and the proteasome inhibitor MG132, the
expression of E7 happened to be remarkably increased, which indicated that E7 might
be degraded through E3 ubiquitin ligases by activating the NEDD8 (Fig.5B).
The top 6 categories, based on IPA (ingenuity pathway analysis) function, including
“cancer, gastrointestinal disease”, “hepatic system disease”, “organismal injury and
abnormalities”, “gene expression”, “organismal injury and abnormalities”, “renal and
urological disease” and so on (Fig.5C). The pathway enrichment analysis was also
showed below. The orange polyline indicated the degree of enrichment (-log (p-value))
of the pathway in the data set. Corresponding pathways in the orange bar graph were
predicted activated pathways; the blue bar graph was predicted inhibited pathways
(osteoarthritis pathway) (Fig.5C); gray pathways were temporarily unable to be
automatically scored for prediction; white pathways had insufficient evidence to
predict their activity and inhibition currently. Osteoarthritis is characterized by
degenerative alterations of articular cartilage including both the degradation of
extracellular matrix and the death of chondrocytes. The PI3K/Akt pathway has been
demonstrated to involve in both processes [22], which was also associated with
several pathways relating the process of cervical carcinogenesis. We proposed that the
compound H1 might be a multi-target inhibitor, acting on a complex pathway network
associated with tumorigenesis. It needs more effort to investigate the specific
mechanism of the anti-cancer effect of compound H1.

Fig5| (A) Volcano plots and analysis of differential abundant proteins. Green
represented down-regulated proteins, red represented up-regulated proteins. (B)
Western blot analysis of E7 expression following treatment of proteasome inhibitor.
(C) Functional analysis of differentially expressed proteins based on enrichment
analysis of GO and pathway.
Conclusions
Epidemiological studies have clearly established HR-HPV infection as the central
cause of invasive cervical cancer [25]. Essential for the normal virus life cycle, the
HPV oncoprotein E7 plays a part throughout the cervical cancer development
pathway [3]. The best-recognized mechanism of gene regulation by E7 is through the
Rb-E2F pathway, which affects the transcriptional process and DNMT1 expression.
Herein, we identified compound H1 as an inhibitor against cervical cancer, with
moderate potency and selectivity. Starting from virtual screening, the
structure-activity study was executed to discover more available compounds. Finally,
compound H1 emerged as the most potent inhibitor, which presented a first-rate
inhibitory activity against cervical cancer in vitro (IC50 = 380 nM) and in vivo.
Compound H1’s anti-cervical effect was associated with the repression of the
oncoprotein E7. Especially, H1 was involved in the E7-Rb-E2F transcriptional
process and DNMT1 relevant cellular pathway, which played an important role in cell
cycle and cell proliferation [23]. We also exploited proteomics analysis. It turned out
that compound H1 might be a multi-target inhibitor, acting on a complex pathway
network associated carcinogenic process. Further study is ongoing in our laboratory to
unravel the biological mechanism of cervical cancer inhibition.
In conclusion, after the virtual screening, the compound structure was derivatized to
study the SARs. Eventually, we discovery the compound with specifically inhibitory

activity of cervical cancer named compound H1. Our results showed that compound
H1 had profound antiproliferative activities against cervical cancer in vitro and in
vivo. Concomitantly, our findings provide strong evidence of compound H1 as a
suppressor against cervical cancer, which is worth the further optimization of this
structure and the development in the pursuit of biological mechanisms.

Experimental section
1
. Virtual screening
Substructure screening was processed on Pipeline Pilot 8.5.
. Compound synthesis and characterization
2
The
screened
compounds
were
purchased
from
SPECS
database
(http://www.specs.net) or PKU-CNCL (http://www.pkucncl.com/). The purities of all
purchased compounds are more than 90%. For the chemical synthesis study, synthesis
reagents and solvents were obtained from J&K Chemical (Beijing, China) and used
1
13
without further purification. H and C NMR spectra were recorded on Bruker (400
MHz) instruments, using DMSO-d or CDCl as solvents. High-resolution mass
6
3
spectra (HRMS) were recorded on a Bruker Apex IV FTMS mass spectrometer using
ESI (electrospray ionization). Purity of the final compounds was determined by NMR
and RP-HPLC to be > 95%.
3
. General procedure for H1 and compound 61 - 63 synthesis.
2
, 4-diaminophenol dihydrochloride (1 mmol) and 2-aminobenzothiazole-6-formic
acid (1 mmol) were add to a round bottom flask. The Eaton’s reagent (2 mL) was
added and refluxed for 8 hours. The solution was diluted in 100 mL water and
neutralized by saturated NaHCO solution, the purple black solid precipitated
3
subsequently. The crude product was obtained after filtering and washing. These
crude products were separated by column chromatography (DCM: MeOH 10:1, a
little ammonia) and finally brown powder H1 was obtained, yield 51%.
2
- (2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine (1, H1). Brown powder,
1
Yield 51%, Mp 277-278℃ . H-NMR (400MHz, DMSO-d ) δ 8.44 (s, 1H), 7.98 (d,
6
J=8.6Hz, 1H), 7.86 (s, 2H), 7.45 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.6Hz, 1H), 6.83 (d,
13
J=1.7 Hz, 1H), 6.62 (dd, J=8.6, 1.9Hz, 1H), 5.08 (s, 2H). C-NMR (101 MHz,
DMSO-d ) δ 169.4 163.0 156.0 146.9 143.3 143.1 132.4 125.3 120.4 119.8 118.3
6
+
+
1
12.9 110.7 103.1. HRMS (ESI-TOF ) m/z calculate for C H N OS [M+H] m/z:
14 11 4
2
83.0654, found: 283.0653.
2
1
8
- (benzo[d]thiazol-6-yl) benzo[d]oxazole (61). Yellow powder, yield 44%, Mp
1
58-159℃ . H-NCMR (400MHz, DMSO-d ) δ 9.59 (s, 1H), 9.09 (d, J = 1.3 Hz, 1H),
6
.36 (dd, J = 8.6, 1.6 Hz, 1H), 8.29 (d, J =8.6 Hz, 1H), 7.86 - 7.82 (m, 2H), 7.49 - 7.41
1
3
(m, 2H). C-NMR (101 MHz, DMSO-d ) δ 162.5, 160.1, 155.5, 150.8, 142.1, 135.2,
6
+
1
26.1, 125.6, 125.5, 124.2, 123.9, 122.8, 120.4, 111.4. HRMS (ESI-TOF ) m/z
+
calculate for C H N OS [M+H] m/z: 253.0436, found: 253.0438.
1
4
9
2
6
- (benzo[d]oxazol-2-yl) benzo[d]thiazol-2-amine (62). Yellow powder, yield 56%,

1
Mp 243-244℃ . H-NMR (400MHz, DMSO-d ) δ 8.52 (s, 1H), 8.05 (d, J = 8.4 Hz,
6
1
H), 7.92 (s, 2H), 7.76 - 7.73 (m, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.42 - 7.34 (m, 2H).
1
3
C-NMR (101 MHz, DMSO-d ) δ 169.7, 163.3, 156.5, 150.6, 142.3, 132.4, 125.6,
6
+
1
25.4, 125.2, 120.8, 119.8, 119.1, 118.3, 111.1. HRMS (ESI-TOF ) m/z calculate for
+
C H N OS [M+H] m/z: 268.0545, found: 268.0546.
14
10
3
2
- (benzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine (63). Yellow powder, yield 49%,
1
Mp 184-185℃ . H-NMR (400MHz, DMSO-d ) δ 9.55 (s, 1H), 8.97 (s, 1H), 8.28 (d, J
6
=
8.6 Hz, 1H), 8.23 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.92 (s, 1H), 6.71 (d,
1
3
J = 8.5 Hz, 1H), 5.18 (s, 2H). C-NMR (101 MHz, DMSO-d ) δ 162.1, 159.6, 155.2,
6
1
47.2, 143.3, 143.1, 135.1, 125.4, 124.5, 124.1, 122.2, 113.8, 111.0, 103.1. HRMS
+
+
(ESI-TOF ) m/z calculate for C H N OS [M+H] m/z: 268.0545, found: 268.0544.
14 10 3
4
. General procedure for compound 64 synthesis.
H1 (30 mg, 0.1 mmol) was dissolved in DMF (1 mL), N, N’-carbonyldiimidazole (17
mg, 0.1 mmol) and Et N (10 mg, 0.1 mmol) were added and reacted at room
3
temperature for 1 hour. Then DMF solution (1 mL) with ethylamine (0.15 mmol) was
added and reacted overnight at room temperature. After the reaction was completed, it
was separated by column chromatography (DCM: MeOH 10:1 - 5:1, a little
ammonia).
1
-(2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-yl)-3-ethylurea (64). White
1
powder, yield 25%, Mp > 320℃ . H-NMR (400MHz, DMSO-d ) δ 8.58 (s, 1H), 8.50
6
(d, J = 1.5 Hz, 1H), 8.03 (dd, J = 8.5, 1.6 Hz, 1H), 7.90 (m, 3H), 7.58 (d, J = 8.7 Hz,
1
3
1
H), 7.48 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 8.8, 1.9 Hz, 1H), 6.12 (t, J = 5.5 Hz, 1H),
1
3
.17 - 3.09 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). C-NMR (101 MHz, DMSO-d ) δ 169.6,
6
63.6, 156.3, 155.8, 145.6, 142.6, 138.3, 132.4, 125.5, 120.7, 119.3, 118.3, 116.2,
+
110.6, 108.4, 34.5, 16.0. HRMS (ESI-TOF ) m/z calculate for C17H16N5O2S
+
[
M+H] m/z: 354.1025, found: 354.1014.
5
. Chemicals and reagents
Antibodies against DNMT1 (#5032), GAPDH (#5174), Rb (#9313), p-Rb (Ser608)
#8147) and p-Rb (Ser780) (#3590) were purchased from Cell Signaling Technology.
(
Antibody for E6 (#ab20192), E7 (#ab100953) and E2F-1 (#ab112580) were
purchased from Abcam. All other chemicals used were of analytical grade.
6
. Cell culture
HeLa, SKOV-3, A375 and HEK293 were purchased from national infrastructure of
cell line resource, which were cultured in DMEM supplemented with 10% fetal
bovine serum (FBS) and with 1% antibiotics (penicillin 100 IUꢀml-1 and streptomycin
1
0ꢀμgꢀml-1). MCF7, A549, U87, NCI-H460 and A431 cells are generous gifts from

the Zhenjun Yang group, which were cultured in RPMI 1640 containing supplements
0% FBS, 1% antibiotics (penicillin 100 IUꢀml-1 and streptomycin 10ꢀμgꢀml-1). Cells
1
were maintained in 5% CO2 incubator at 37°C. Experimental consumables were
purchased from Wuxi NEST Biotechnology Co., Ltd. (Wuxi, China).
7
. Cell proliferation assay
The effect of H1 on cell viability was determined by the SRB (Sulforhodamine B)
assay as described previously [26]. Briefly, cells were cultured on sterile slides in
9
6-well plates overnight at 37 °C with 5 % CO2. Then cells were treated with various
dosages of compounds or dimethyl sulfoxide (DMSO) (vehicle) for 72h, 200μl 10%
trichloroacetic acid (TCA) was added to each well, followed by incubation for 1h at
4
°C in a refrigerator. After the microplate was washed five times with sterile water,
evaporation in air, 100 μl of 0.4% SRB dissolved in 1% acetic acid was added to each
well. Following an additional incubation at room temperature in the dark for 15
minutes. After the microplate was washed five times with 1% acetic acid, evaporation
in air, add 100μl 10mmol/L Tris-base to each well to dissolve SRB for 30 minutes.
Absorbance was measured at 540 nm using a microplate reader.
8
. Flow cytometry assays.
Cells were washing with PBS before gathered and fixed with 70% ethanol at 4 °C
overnight. After two rinses with PBS, Cells were then incubated with RNase and
propidium iodide as per the manufacturer’s instructions (Beyotime). DNA was
observed using flow cytometry (Beckman CytoFlex) to calculate the percentage of
cells in G0/G1, S and G2/M phases with Novo Express software. A cell apoptosis
assay was performed using according to the manufacturer’s instructions (Beyotime).
Briefly, treated cells were washed with PBS twice and then cells were resuspended in
5
00μl of binding buffer.1μl of FITC-labelled Annexin V and propidium iodide were
added to samples and incubated in the dark for 15ꢀmin at room temperature. Cells
were subjected to FACS analysis within 1 hour [27].
9
. Western blotting
Cells were collected and lysed in prechilled RIPA buffer (50mM Tris-HCl at pH8.0,
50mM sodium chloride, 0.1% SDS, 0.5% DOC, 1% NP-40 and 1mM phenyl methyl
1
sulphonyl fluoride (PMSF) for 10min on ice, shake for 30s every five minutes. Then
repeated freeze-thaw 3 times in liquid nitrogen and 37℃ water baths. Homogenates
were centrifuged for 30min at 4 °C and the supernatants were collected and boiled in
SDS loading buffer. Proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and then western blotting was
performed as described previously [28]. Sample (35 μg) was loaded onto a 10% SDS–
polyacrylamide gel electrophoresis (PAGE) gel and protein was transferred to a
polyvinylidene fluoride (PVDF) membranes for antibody detection. Then blocked

with 5% non-fat milk powder in TBST buffer for 1 h at RT. The membrane was then
incubated with a diluted primary antibody in blocking buffer overnight at 4 °C on a
shaker. The membrane was washed three times with TBST and then incubated with a
secondary antibody for 1 hour at room temperature. Protein bands on western blots
were visualized by chemiluminescent detection (ECL, Amersham).
1
0. Xenograft Cervical Cancer Models
Male BALB/c nude mice of 4-week-old were purchased from the department of
6
laboratory animal science (Beijing, China). HeLa cells (1*10 in 200ul PBS) were
3
inoculated subcutaneously on the flanks. When the tumor size reached ∼50 mm , the
mice were randomly divided into four groups, receiving treatment with placebo (PBS,
i.g., alternative day), compound H1 (50ꢀmg/kg, i.g., alternative day), respectively.
Tumors were measured alternate days with calipers and tumor volumes were
2
ꢀ
calculated by the following formula: a ꢀ×ꢀbꢀ×ꢀꢁ, where “a” is the smallest diameter and
“b” is the diameter perpendicular to “a”. After mice were euthanized, xenografts were
dissected, weighed, and preserved.
1
1. Protein Digestion
HeLa cell lysate was collected after treating with compound H1 at a concentration of
0μM for 6 hours. The filter-aided sample preparation (FASP) method is used for the
1
digestion[29], and all buffer exchanges were carried out by centrifugation at 14, 000g
for 15 min. Protein extract (300 μg) was transferred into a vivacon 500 filtrate tube
(10, 000 MWCO, Sartorius Stedim Biotech). The buffer was exchanged with 200μL
of 8 M urea in 50mM NH HCO for three times. The cysteine bonds were reduced by
4
3
5
0μL Tris (2-carboxyethyl) phosphine (10 mM, TCEP; Abcam, Cambridge, MA, USA)
solution, and the alkylation was carried out following the incubation in the dark for 30
min with iodacetamide. 200μL of 50 mM NH HCO were used to remove the
4
3
reducing and alkylating agents for three times. 100 μL of 50 mM NH HCO
4
3
containing 1:50 ratio of sequencing-grade modified trypsin (V5111; Promega,
Fitchburg, WI, USA) to volume of protein was added to the filters. The trypsin
digestion was incubated at 37℃ overnight. 50 μL of 50 mM NH HCO was used to
4
3
elute the peptide-rich solution for three times.
1
2. Peptides separation
Peptides separation was performed with the Dionex Ultimate 3000 system. Peptide
mixture was first loaded onto a trap column and eluted by mobile phase A (0.1%
ammonium hydroxide solution). Then, peptides separation was carried out on a C18
column (VBridgeTM BEH130, Waters) at a flow rate of 230μL/min using a gradient
of 5% phase B (0.1% ammonium hydroxide in 80% acetonitrile) for 5 min, 5 to 18%
phase B for 4 min, 18 to 20% phase B for 10 min, 20 to 22% phase B for 6 min, 22 to
2
7% phase B for 8 min, 27 to 36% phase B for 6 min, 36 to 43% phase B for 4 min,

4
3 to 100% phase B for 3 min, and maintain 100% phase B for 20 min. A total of 10
fractions were collected, dried by vacuum, and ready for nano LC-MS/MS analyses.
1
3. Data analysis
Raw files were analyzed using MaxQuant_1.5.1.2. The database search was
performed in the Uniprot human protein database using the Andromeda search engine.
Trypsin was chosen and a maximum of two missed cleavages was allowed,
carbamidomethylation of Cys was set as fixed modifications, and oxidation of Met
was set as variable modifications. Label-free quantitation (LFQ) was selected. Other
settings were default. Perseus v 1.5.2.6 software was used for quality control and
statistical processing[30]. All potential contaminants, reverse hits and hits only
identified by site were filtered out prior to log2 transformation of the LFQ
intensities[31].
Abbreviation
HR-HPV, high-risk HPV; PKU-CNCL, Chinese National Compound Library of
Peking University; SRB, Sulforhodamine B; Rb, retinoblastoma protein; DNMT,
DNA methyltransferase; IC , half-maximal inhibitory concentration; FCM analysis,
5
0
Flow cytometry analysis.
Acknowledgments
This research was supported by the National Natural Science Foundation of China
21772005, 81872730) and the Beijing Natural Science Foundation (7172118). We are
(
very appreciative of Dr. Wei Huang (Chinese Academy of Medical Sciences, Beijing),
Dr. Bo Xu (State Key Laboratory of Natural and Biomimetic Drugs, Beijing) and Lan
Yuan (Peking University, Beijing) for their help in this work.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the
content and writing of this article.
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(

Highlights:
ó Identification of 2-(2-aminobenzo[d]thiazol-6-yl) benzo[d]oxazol-5-amine
derivatives and compound H1 was identified as a specific inhibitor for
HPV18-positive cervical cancer cells.
ó The compound H1 inhibited expression of HPV E7 protein which associated with
cell proliferation process, preventing cervical cancer cells from premature cell
procession and abnormal proliferation.
ó E7-Rb-E2F cellular pathway and DNA methylation were suppressed by compound
H1.
ó Proteomics analysis revealed that E7 might be degraded through E3 ubiquitin
ligases, which aligned with decreasing expression of E7 following the treatment of
compound H1.

Declaration of interests
☒
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.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: