Synthesis and biological evaluation of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-ones as cisplatin sensitizers

Article abstract of DOI:10.1039/c8md00464a

A series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized and their anti-cancer as well as cisplatin sensitization activities were evaluated. Among them, compounds 6e and 6h exhibited significant cisplatin sensitization activity against HCT116. Hoechst staining and annexin V-FITC/PI dual-labeling studies demonstrated that the combination of 6e/6h and cisplatin can induce tumour cell apoptosis. Western blot showed that the expression of ATR downstream protein, CHK1, decreased in 6e + cisplatin and 6h + cisplatin groups compared with that in the test compound and cisplatin group. Furthermore, docking of 6e/6h into the ATR structure active site revealed that the N1 and N8 atoms in the naphthyridine ring and the hybrid atom in the oxadiazole ring are involved in hydrogen bonding with Val170, Glu168 and Tyr155. Additionally, the naphthyridine ring is also involved in π-π stacking with Trp169. Accordingly, compounds 6e and 6h can be expected to be potential cisplatin sensitizers that can participate in HCT116 cancer therapy.

Full text of DOI:10.1039/c8md00464a

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. Hou, H. Luo, M.
Zhang, G. Yan, C. Pu, S. Lan and R. Li, Med. Chem. Commun., 2018, DOI: 10.1039/C8MD00464A.
This is an Accepted Manuscript, which has been through the
Volume 7 Number 1 January 2016 Pages 1–204
Royal Society of Chemistry peer review process and has been
accepted for publication.
MedChemComm
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Broadening the field of opportunity for medicinal chemists
www.rsc.org/medchemcomm
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
Themed issue: Antibiotic Resistance
ISSN 2040-2503
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
rsc.li/medchemcomm

Please do not adjust margins
MedChemComm
Page 1 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
Synthesis and biological evaluation of 3-(1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-ones as cisplatin sensitizers
Received 00th January 20xx,
Accepted 00th January 20xx
Xueyan Hou,a,b,# Hao Luo,a,# Mengqi Zhang,a Guoyi Yan,a Chunlan Pu,a Suke Lan,a and Rui Lia,*
DOI: 10.1039/x0xx00000x
A series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized and their anti-cancer as well as cisplatin
sensitization activities were evaluated. Among them, compound 6e and 6h exhibited the significantly cisplatin sensitization activity against
HCT116. Hoechst staining and annexin V-FITC/PI dual-labeling studies demonstrated that the combination of 6e/6h and cisplatin can induce
tumour cells apoptosis. Western blot showed the expression of ATR downstream protein, CHK1, decreased in 6e + cisplatin and 6h +
cisplatin groups, compared with that in test compound and cisplatin group. Furthermore, docking of 6e/6h into the ATR structure active site
revealed that the N1 and N8 atoms in the naphthyridine ring and the hybrid atom in the oxadiazol ring are involved in hydrogen bonding
with Val170, Glu168 and Tyr155. Additionally, the naphthyridine ring is also involved in π-π stack with Trp169. Accordingly, compounds 6e
and 6h can be expected to be the potential cisplatin sensitizers to participate in HCT116 cancer therapy.
www.rsc.org/
structure of target compounds in this study.
Introduction
1,8-naphthyridinone moiety is
a structural feature of many drug
O
CH3
N
O
molecules and pharmaceutical agents and lots of compounds bearing 1,8-
naphthyridinone exhibit a variety of biological properties, such as anti-
tumour,1-3 anti-bacterial,4-5 anti-inflammatory,6 CB2 selective agonist
activities,7-10 etc. As far as anti-cancer was concerned, Ke Chen and his co-
workers discovered a series of 2-aryl-1,8-naphthyridin-4(1H)-ones and 2-
phenyl-1,8-naphthyridin-4-ones as anti-tumour agents through inhibiting
tubulin polymerization.2-3 Indeed, HKL-1 (2-(3-Methoxyphenyl)-5-methyl-1,8-
naphthyridin-4(1H)-one) was a antimitotic agent that can induce G2/M
arrest and mitotic catastrophe in human leukemia HL-60 cells.1
N
N
H
N
H
OCH3
HKL-1 1-2
Com.d 44 3
N
O
Furthermore, 1,3,4-oxadiazole is
a
commonly used moiety for
N
pharmacophore development, which has been thoroughly investigated
because of its hydrogen bonding ability within the receptor site and good
metabolic profile.11 The azole group (―N=C―O) of 1,3,4-oxadiazole can
increase the lipophilicity of drugs, promote drug’s transportation through
cell membranes to reach the target site, and facilitate drug’s various
biological activities.12 In fact, a lot of researches showed that the synthetic
compounds bear 1,3,4-oxadiazole moiety have excellent anticancer
activities both in vitro and in vivo.13-19 Although various substituent 1,8-
naphthyridinones and 1,3,4-oxadiazole have been developed, there are still
a lot of analogues have not been designed and synthesized. Therefore, a
novel molecular skeleton, 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-
one (Fig. 2), was designed and synthesized, which was seen as the core
N
N
H
O
N
O
O
N
N
N
S NH
Cl
O
ZD4054 13
5h 18
Fig. 1. Chemical structures of small molecule compounds bearing 1,8-
naphthyridin-4(1H)-one or oxadiazole with anti-cancer activities.
O
O
N
N
N
N
H
Fig. 2. Chemical structure of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-
one.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 11
View Article Online
DOI: 10.1039/C8MD00464A
ARTICLE
Journal Name
Cisplatin is a DNA-damaging agent that commonly used in the treatment
of solid tumours, which are highly effective against certain cancers like
metastatic testicular cancer.20-23 But for the majority of solid tumours such
as ovarian, lung, and prostatic tumours, cisplatin only has modest benefit.24
Additionally, the drug resistance, nephrotoxicity, ototoxicity and
ATR/CHK1 pathway, which activated by single-stranded DNA arising from
stalled replication forks caused by replication stress or as an intermediated
of DNA repair processes.34-35 Many studies showed that the cisplatin
sensitivity activities can increase by the inhibition of ATR and its
downstream protein CHk1.36-38
neurotoxicity of cisplatin also limit the scope of its application.16 Recent
researches demonstrate that the poor response to those cancers and drug
resistance are closely related to the DNA damage response (DDR).26-31 DDR
In this study, a series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-
4(1H)-one derivatives were designed and synthesized (Scheme 1 and 2), and
their biological activities were evaluated as anti-cancer and cisplatin
network is an elaborate transduction system which can sense different types sensitizer agents. Among of these compounds, 6e and 6h were discovered
of damage and coordinate a series of response, including activation of
apoptosis, cell cycle control, transcription, senescence, and DNA repair
processes.32-33 One critical signalling pathway in DDR network is the
the cisplatin sensitization effects. Hoechst staining, annexin V-FITC/PI dual-
labelling, western blot and molecular docking experiments were employed
to investigate the possible mechanism.
Scheme 1. Reagents and conditions: (i) diethyl 2-(ethoxymethylene)malonate, 130 °C, 2 h; (ii) Ph2O, 250 °C, 30-40 min; (iii) (4-(methylsulfonyl)phenyl)boronic
acid, Pd Cat., 60 °C, 8 h; (iv) H2NNH2, 50 °C, 3 h; (v) RCOCl, THF, DIEA, overnight; (vi) pyridine, SOCl2, 3 h.
Cl
N
N
O
N
O
N
O
O
H
N
iii
iv
Br
R1
Cl
Cl
Br
ii
O
O
N
H
O
N
N
H
N
N
H
N
N
H
O
O
O
O
10
8
9
Br
Br
NH2
O
N
10a: R1= -pyrid-3-yl
10b: R1= -C6H4-4-CN
10c: R1= -C6H5
H
i
N
N
H
N
N
H
2
7
F
N
N
O
N
O
N
O
O
H
N
vi
vii
Br
R2
F
F
Br
v
O
O
N
H
O
N
N
H
N
N
H
N
N
H
13
11
12
13a: R2= -pyrid-3-yl
13b: R2= -pyrid-4-yl
13c: R2= -C6H4-4-CN
Scheme 2. Reagents and conditions: (i) H2NNH2, 50 °C, 3 h; (ii) R1COCl, THF, DIEA, overnight; (iii) pyridine, SOCl2, 3 h; (iv) aryl boronic acid or aryl borate, Pd
Cat., 50 °C, 6 h; (v) R2COCl, THF, DIEA, overnight; (vi) pyridine, SOCl2, 3 h; (vii) aryl boronic acid or aryl borate, Pd Cat., 50 °C, 6 h.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 3 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
6h, respectively. However, the cytotoxicity against LO2 normal cells did not
significantly increase in 6e/6h + cisplatin group (Table S2 of the ESI).
Results and discussion
Chemistry
In this study, a new series of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-
4(1H)-one derivatives were synthesized. The general procedures for the
preparation of the 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one
derivatives 6a-6h were efficiently synthesized according to the protocol
outlined in Scheme 1. Condensation of 5-bromopyridin-2-amine with diethyl
ethoxymethylenemalonate were heated under 130 °C for 2 h to provide the
intermediate 1. Phenyl ether was heated under stirring at 250 °C, and then 1
was added slowly. The resulting mixture was refluxed for 4 h to get the
intermediate 2. Intermediate 3 was prepared from 2 by Suzuki coupling with
Morphological analysis by Hoechst staining
We next explored the morphology of cancer cell nuclei by Hoechst 33258
staining. Hoechst 33258 is a membrane-permeable fluorescent dye that can
stain the chromatin of cells. In cells with normal morphology, the nuclei
stained by Hoechst 33258 were round and light-blue coloured, and the
chromatin was stained blue. While the apoptotic cells have condensed
chromatins, bright blue nuclei, and nuclear fragmentation.40-41 In this study,
the treatment of cisplatin, 6e or 6h alone can induce morphological changes
of HCT116 nuclei. In fact, the morphology of cancer cell nuclei could
undergo further changes such as remarkable chromatin condensation, cell
shrinkage, and evident reduction in the number of adherent cells (Fig. 3a).
The number of nuclei were quantified and analyzed that shown in Fig. 3b,
which was consistent with MTT assay.
4-(methylsulfonyl)phenylboronic
acid
and
dichlorobis(triphenylphosphine)palladium(II). Intermediate 4 was obtained
by treating 3 with hydrazine hydrate in the presence of methanol at room
temperature. A series of aromatic acid derivatives were reacted with SOCl2
and DMF as catalyst refluxed for 1 h to afford aroyl chloride derivatives,
which were converted to amide intermediates 5a-5h by the treatment with
4 in the presence of DIEA. Finally, the compounds 6a-6h were accomplished
by refluxing 5a-5h with SOCl2 in pyridine for 3 h.
Apoptosis analysis by FCM
To further understand the morphological changes of cancer cells, Annexin V-
FITC/PI dual-labelling was performed by FCM. As shown in Fig. 4a, cisplatin,
6e and 6h at different concentrations can induce HCT116 cell apoptosis at
different degrees. Remarkably, the percentage of Annexin V-positive cells
significantly increase in the 6e + cisplatin group, which indicated that
cisplatin and 6e could produce synergistic apoptosis in the appropriate
concentration (Fig. 4b). At the same time, the percentage of PI-positive cells
was enhanced remarkably (p<0.001) compared with that with cisplatin or 6e
alone (Fig. 4c). Similar results were also observed in 6h + cisplatin group.
Together with Hoechst 33258 staining experiment, we propose that
apoptosis may be one signalling pathway for the enhanced cisplatin
sensitization effects of compounds 6e and 6h.
As outlined in Scheme 2, compounds 10a-10c and 13a-13c were achieved
starting from intermediate 2 and the synthesis began with hydrazine
hydrate to prepare intermediate 7. 8 and 11 were detected by the reaction
of the corresponding aromatic acid derivatives with 7. Refluxing 8 and 11
with SOCl2 in pyridine for 3 h can provide the intermediate 9 and 12.
Bis(triphenylphosphine)palladium(II) chloride catalyzed coupling of 9 with a
series of boronic acid derivatives in the presence of Na2CO3 to yield the
compounds 10a-10c. In addition, compounds 13a-13c were obtained by the
similar synthetic route with intermediate 12 and different kinds of boronic
acid analogues. All synthetic compounds gave satisfactory analytical and
spectroscopic data, which were consistent with their depicted structures.
Western blot analysis
In vitro inhibition evaluation of compounds
The cisplatin sensitization mechanism initiated by 6e/6h in combination
with cisplatin was explored by western blot. The ATR/CHK1 signalling
pathway can regulate many common proteins, such as p53, CDC25
phosphatases, and Wee1 kinase, which further regulated G1, S and G2/M
checkpoints. Besides, suppresses of CHK1 signalling pathway will lead to
DDR damage and then may trigger DNA damage-induced apoptosis or
senescence.42 It has been reported that some small molecule compounds,
such as SCH900776 (MK-8776)36 and CBP-9387239, can enhance the cisplatin
sensitivity by inhibiting phosphorylation of CHK1, and induce cell death.
Therefore, the expression of CHK1 in HCT116 cancer cells was examined
in this study. The results showed that phosphorylated CHK1 was
upregulated by cisplatin treatment (Fig. 5), which may be attributed to the
DNA-damage agent cisplatin, leading to the DDR in cancer cells. However,
the CHK1 expression was decreased in 6e + cisplatin and 6h + cisplatin
groups with a concentration-dependent manner, indicating that 6e and 6h
can inactivate the CHK1 induced by cisplatin treatment and then achieve the
synergistic anti-tumour effects. The expression of CHK1 can recover to
normal in 6e (2.5 μM)
The proliferation inhibitory effects of compounds 6a-6h, 10a-10c and 13a-
13c in several tumour cell lines were evaluated primarily by MTT. It was
shown that the cancer cells, including human non-small cell lung cancer
A549, hepatocellular carcinoma HepG2, oral epidermoid carcinoma KB,
breast cancer MCF-7, cervical carcinoma Hela and colorectal carcinoma
HCT116, were inhibited by the treatment of each compound with different
concentrations (Table 1 and 2). However, compared with that of cisplatin,
the inhibitory effects of compounds were slight. Comfortingly, all
compounds have lower cytotoxicity to LO2 normal cells (Table S1 of the ESI).
In vitro inhibition evaluation of compounds in combination with cisplatin
To investigate whether compounds 6a-6h, 10a-10c and 13a-13c have
activities in tumour therapy, the combination effects of each compound and
cisplatin against HCT116 cells were evaluated in this study. As shown in
Table 3, the inhibitory rate of cisplatin (2.5 μM) was 26.98%, while the
inhibition rate of each compound (2.5 μM) was low. It is worthy to note that
the compounds, especially 6e and 6h, can enhance the sensitivity of tumour
cells to cisplatin (p＜0.01 or p＜0.001). The inhibition rate of cisplatin
increased from 26.98% to 50.81% and 54.78%, after combining with 6e and
+ cisplatin and 6h (5 μM) + cisplatin groups.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 11
View Article Online
DOI: 10.1039/C8MD00464A
ARTICLE
Journal Name
Table 1. In vitro cancer cell growth inhibiting rate of compound (10 μM) alone.
Inhibition rate (% of control)
Compound
(10 μM)
A549
HepG2
KB
MCF-7
Hela
HCT116
6a
6b
9.51 ± 1.38
6.98 ± 1.67
5.20 ± 1.80
2.83 ± 1.11
17.53 ± 0.25
15.71 ± 1.13
14.73 ± 0.59
7.83 ± 2.58
16.22 ± 1.46
25.19 ± 0.65
17.00 ± 2.69
18.65 ± 1.19
24.44 ± 0.69
11.83 ± 2.71
18.30 ± 2.66
21.63 ± 2.40
19.68 ± 1.54
20.53 ± 2.07
18.89 ± 0.77
57.76 ± 1.09
-
-
-
-
-
6c
12.11 ± 0.50
6.65 ± 2.10
3.02 ± 0.21
4.27 ± 0.44
18.36 ± 1.02
6d
-
-
-
-
-
6e
8.95 ± 1.95
13.34 ± 2.12
7.50 ± 2.19
7.76 ± 0.67
2.57 ± 0.38
6f
26.76 ± 0.90
11.61 ± 2.75
9.30 ± 0.66
13.07 ± 0.38
1.64 ± 0.76
6g
3.59 ± 0.61
11.09 ± 2.52
7.58 ± 2.62
8.10 ± 0.97
15.68 ± 0.10
6h
11.22 ± 0.95
8.94 ± 1.91
3.19 ± 1.41
10.58 ± 1.44
18.48 ± 1.25
10a
10b
10c
13a
13b
13c
Cisplatin
-
-
-
-
-
12.51 ± 0.78
12.67 ± 1.84
9.73 ± 3.29
19.69 ± 0.56
13.65 ± 0.43
9.77 ± 1.63
14.93 ± 2.31
19.49 ± 2.88
26.90 ± 0.30
31.29 ± 0.18
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
68.52 ± 0.53
76.16 ± 3.94
42.43 ± 3.58
27.37 ± 0.56
44.16 ± 0.56
Table 2. In vitro cancer cell growth inhibiting rate of compound (5 μM) alone.
Inhibition rate (% of control)
Compound
(5 μM)
A549
HepG2
KB
MCF-7
Hela
HCT116
6a
6b
8.17 ± 0.50
5.46 ± 0.22
4.38 ± 2.73
1.38 ± 0.65
8.58 ± 0.34
8.60 ± 2.16
9.26 ± 1.42
5.61 ± 1.32
11.34 ± 3.27
20.35 ± 1.25
16.38 ± 1.25
14.86 ± 2.89
20.44 ± 1.46
15.86 ± 1.02
17.86 ± 2.56
12.29 ± 2.97
11.09 ± 0.24
10.93 ± 2.17
12.56 ± 1.84
41.12 ± 0.39
-
-
-
-
-
6c
6.76 ± 2.01
3.49 ± 0.30
2.16 ± 0.81
2.35 ± 0.35
7.08 ± 0.04
6d
-
-
-
-
-
6e
7.92 ± 2.15
13.20 ± 1.76
3.70 ± 0.81
1.86 ± 0.36
1.49 ± 0.48
6f
23.77 ± 0.21
11.25 ± 3.26
2.15 ± 2.10
0.86 ± 0.10
1.07 ± 0.57
6g
3.11 ± 0.15
9.45 ± 2.84
2.55 ± 3.38
3.78 ± 0.50
8.47 ± 1.71
6h
6.69 ± 0.67
6.90 ± 1.65
0.43 ± 0.60
6.26 ± 0.30
10.45 ± 0.34
10a
10b
10c
13a
13b
13c
Cisplatin
-
-
-
-
-
7.60 ± 0.99
11.51 ± 4.45
7.35 ± 0.81
4.65 ± 3.23
4.80 ± 0.58
1.44 ± 0.85
5.10 ± 1.20
2.15 ± 1.06
11.95 ± 0.42
3.55 ± 1.58
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
39.77 ± 1.01
55.09 ± 3.21
24.30 ± 3.29
7.76 ± 1.35
34.99 ± 0.16
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 5 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
Table 3. In vitro cancer cell growth inhibiting rate of compound combined with cisplatin for HCT116 cells.
Inhibition rate (% of control)
Cisplatin
(2.5 μM)
Compound
(2.5 μM)
Cisplatin (2.5 μM) +
Compound (2.5 μM)
26.98 ± 2.84
6a
6b
0.53 ± 2.17
4.32 ± 0.88
2.64 ± 1.71
5.37 ± 1.71
8.13 ± 3.63
2.06 ± 3.38
10.71 ± 1.29
7.47 ± 0.73
10.42 ± 2.08
13.73 ± 3.16
9.81 ± 5.15
9.87 ± 2.03
7.54 ± 1.26
10.15 ± 0.27
27.19 ± 2.16
28.59 ± 1.63
29.16 ± 1.88
34.28 ± 2.56
50.81 ± 1.35***###
24.91 ± 2.06
39.46 ± 0.75
54.78 ± 2.70***###
20.13 ± 1.37
35.43 ± 1.99
29.83 ± 0.36
20.06 ± 2.12
19.57 ± 0.29
25.64 ± 1.82
6c
6d
6e
6f
6g
6h
10a
10b
10c
13a
13b
13c
*p<0.05, **p<0.01 and ***p<0.001 vs. cisplatin group; #p<0.05, ##p<0.01 and ###p<0.001 vs. compound group.
Fig. 3. Fluorescence microscopic appearance of Hoechst 33258-stained nuclei of HCT116 cells with the treatment of compounds 6e and 6h in combination
with cisplatin. HCT116 cells were treated with 6e or 6h alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. (a) i, control; ii, cisplatin (2.5 μM);
iii, 6e (2.5 μM); iv, 6e (2.5 μM) + cisplatin (2.5 μM); v, 6h (2.5 μM); vi, 6h (2.5 μM) + cisplatin (2.5 μM). (b) The blue fluorescence was quantified and analyzed
by T test. All data were from three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs. cisplatin group; #p<0.05, ##p<0.01 and ###p<0.001 vs.
compound group.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 11
View Article Online
DOI: 10.1039/C8MD00464A
ARTICLE
Journal Name
Fig. 4. Apoptosis analysis of compounds 6e and 6h in combination with cisplatin. HCT116 cells were treated with 6e/6h alone for 24 h before combination
with cisplatin for 48 h. Cell apoptosis were evaluated by flow cytometry. (a) i, control; ii, cisplatin (2.5 μM); iii, 6e (2.5 μM); iv, 6e (2.5 μM) + cisplatin (2.5
μM); v, 6h (2.5 μM); vi, 6h (2.5 μM) + cisplatin (2.5 μM). (b and c) Percentage of Annexin V+ and PI+ cells analyzed of 6e and 6h. All data were from three
independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs. cisplatin group; #p<0.05, ##p<0.01 and ###p<0.001 vs. compound group.
Fig. 5. Compounds 6e (a) and 6h (b) in combination with cisplatin inhibited Chk1 expression. HCT116 cells were treated with 6e or 6h alone for 24 h before
combination with cisplatin (2.5 μM) for 48 h. CHK1 expression was quantified by the densitometry analysis using ImageJ. *p<0.05, **p<0.01 and ***p<0.001
vs. cisplatin group.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 7 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
Binding mode of 6e and 6h into ATR
Based on the above western blot result, we suspected that the 6e and
6h might interact with ATR signaling pathway. Therefore, in an attempt to
explain the cisplatin sensitization effects of 6e and 6h, the molecular
docking to ATR43 was performed in our research and the results showed that
the N1 and N8 in the naphthyridine ring and the hybrid atoms in the
oxadiazol ring are involved in H-bond interactions with Val170, Glu168 and
Tyr155 (Figure 6). Additionally, the naphthyridine ring is also involved in π-π
stack with Trp169. Besides the key binding of the skeleton mentioned
above, the sulfone group in R2 also forms the hydrogen bond with Gly175.
Moreover, the p-methylphenyl ring in R1 is involved in hydrophobic
interaction with Lys117, Leu122 and Pro159.
Biological activities of the tested compounds could be correlated to
structure variations. In view of 6a-6h, it was found that the replacement of
phenyl with pyridine group resulted in the decrease in the sensitization
activity, which can be ascribed to the decline of hydrophobic interaction
with Lys117, Leu122 and Pro159 of ATR kinase pocket. Furthermore,
exploration of the impact of the substitution on the different position of the
phenyl group demonstrated that 4-modification is more beneficial than 3-
position (6e and 6h). With regard to 4- substituted compound counterparts
in 6f, 6g, and 6h, the sensitivity activities were decreased in the order of -
CH3 > -Cl > -F, hinting that the appropriate electron density is significant for
the activity. Additionally, 4-(methylsulfonyl)benzene were converted to two
different substitutions to explore the influence of the substituted group at
6-position on activity. However, the sensitivities against HCT116 was almost
lost, which can be attributed to the absent of H-bond interaction between
the sulfone group and Gly175.
Fig. 6. Theoretical binding mode of 6e (a) and 6h (b) to ATR active
binding pocket.
6h + cisplatin groups, suggesting that 6e and 6h can inhibit the DDR
response, and increase the sensitivity of tumour cells to cisplatin.
Furthermore, molecular docking was employed in our study and
Conclusions
docking of 6e/6h into the ATR structure active site revealed that the
In summary, a series of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-
one derivatives were synthesized and their anti-tumour activities in vitro
against A549, HepG2, KB, MCF-7, Hela, and HCT116 cells were tested in this
research. Regrettably, all compounds have only slight activities. However,
the anti-tumour activities initiated by compounds in combination with
cisplatin were measured and the results showed that compound 6e and 6h
bearing the good cisplatin sensitization. Hoechst staining and annexin V-
FITC/PI dual-labeling studies demonstrated that the combination of 6e/6h
(2.5 μM) and cisplatin (2.5 μM) can significantly induce apoptosis of HCT116
cancer cells.
N1 and N8 atoms in the naphthyridine ring and the hybrid atom in the
oxadiazol ring are involved in hydrogen bonding with Val170, Glu168
and Tyr155. Additionally, the naphthyridine ring is also involved in π-π
stack with Trp169. Taken together, 3-(1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one derivatives 6e and 6h were discovered and
deserved further investigation as potential cisplatin sensitizing agents
for HCT116 cancer cells.
Conflicts of interest
ATR is the most important protein in DDR network and western blot
displayed that cisplatin treatment lead to the significant upregulating
of CHK1 expression in HCT116 cancer cells, which indicated that the
DNA-damage agent cisplatin treatment can cause HCT116 cells to
initiate DDR, produce drug resistance and ultimately induce parts of
The authors declare no conflict of interest.
Acknowledgements
We gratefully acknowledge the support from the National Key
Program of China during the 12th Five-Year Plan Period (Grant
tumour cell survival. While the CHK1 expression was decreased in 6e
+
cisplatin and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 8 of 11
View Article Online
DOI: 10.1039/C8MD00464A
ARTICLE
Journal Name
2012ZX09103101-022) and the National Natural Science Foundation of operating at a field strength of 400 MHz at room temperature with TMS and
China (81472780 and 81773195).
solvent signals allotted as internal standards. Chemical shifts are reported in
ppm (δ) with references. Standard and peak multiplicities are designated as
follows: s, singlet; d, doublet; dd, double doublets; t, triplet; q, quartet; brs,
broad singlet; and m; multiplet. Mass spectrometry (MS) data were
obtained by ESI-QTOF spectrometer. Purity of the all compounds was
evaluated by a HPLC system (Dionex Ultimate 3000, USA) was found to be
higher than 98%.
Experimental
Materials and instruments
Unless specified otherwise, all chemicals and reagents used in this study
were of analytical grade and use without any purification. The reactions
were monitored by thin layer chromatography (TLC) on pre-coated silica
GF254 plates (Qingdao Ocean Chemical Factory, China). Melting points
(uncorrected) were determined with a SGW X-4 melting points apparatus
Synthesis of diethyl 2-(((5-bromopyridin-2-yl)amino)methylene)malonate
(1)
Condensation of 5-bromopyridin-2-amine (1.73 g, 10 mmol) with diethyl
ethoxymethylene-malonate (2.16 g, 10 mmol) were heated
(Shanghai precision
& scientific instrument Co., Ltd, China). Magnetic
resonance spectra (NMR) were detected on a Bruker AC-E400 spectrometer
under 130 °C in an open system for 2 h. Then cooled down to room (s, 1H), 8.61 (dd, J = 9.1 Hz, J = 1.7 Hz, 1H), 8.14 (q, J = 8.4 Hz, 4H), 8.03 (d, J =
temperate followed by recrystallization in ethanol to get yellow solid 1 (2.6 9.1 Hz, 1H), 4.71 (s, 2H), 3.31 (s, 3H).
g). Yield: 75.7%. 1H NMR (400 MHz, CDCl3) δ = 11.12 (d, J = 12.5 Hz, 1H), 9.07
Synthesis of compounds 5a-5h
(d, J = 12.7 Hz, 1H), 8.39 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 8.6 Hz, J = 2.4 Hz,
1H), 6.78 (d, J = 8.6 Hz, 1H), 4.50-4.12 (m, 4H), 1.36 dt, J = 15.9 Hz, J = 7.1 Hz,
Appropriate aromatic acid (0.63 mmol) was reacted with SOCl2 (4 mL) and
DMF as catalyst refluxed for 1 h to prepare acid chlorides. The product was
used as this in the next step. A solution of 4 (150 mg, 0.42 mmol) and DIEA
(162 mg, 1.26 mmol) in THF (3 mL) was added to a stirring solution of acid
chlorides in 30 mL of THF at 0 °C. The mixture was stirred at room
6H).
Synthesis of ethyl 6-bromo-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carboxylate (2)
Phenyl ether (30 mL) was heated under stirring at 250 °C. Diethyl 2-(((5- temperature overnight. The white precipitate was recovered by filtration,
bromopyridin-2-yl)amino)methylene)malonate (10g, 29.1 mmol) was slowly washed three times with 5 mL portions of THF and dried overnight under
added, and the resulting mixture was refluxed for 30-40 min. After the vacuum.
mixture was cooled at room temperature, the resulting precipitate was
General procedure for the synthesis of compounds 6a-6h
collected by filtration, washed with petroleum ether, and recrystallized from
DMF to get the white solid 2 (7.8 g). Yield: 90.0%. 1H NMR (400 MHz, CDCl3)
To a mixture of the appropriate intermediate (5a-5h, 0.32 mmol) in SOCl2
δ = 9.38 (d, J = 1.4 Hz, 1H), 9.04 (s, 1H), 7.97 (dd, J = 9.3 Hz, J = 2.0 Hz, 1H),
(5 mL), pyridine (50 mg, 0.64 mmol) was added dropwise. The reaction
7.67 (d, J = 9.3 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H).
mixture was then gradually heated to the reflux temperature and
maintained there for 3 h. The remaining SOCl2 was removed by rotary
Synthesis of ethyl 6-(4-(methylsulfonyl)phenyl)-4-oxo-1,4-dihydro-1,8-
evaporation and poured into saturated NaHCO3 solution to acidify to pH 7.
naphthyridine-3-carboxylate (3)
Then the aqueous layer was extracted with CH2Cl2, and the combined
4-(Methylsulfonyl)phenylboronic acid (2.53 g, 12.65 mmol) was added to organic layers were dried over Na2SO4, filtered and concentrated. The
a solution of intermediate 2 (10.54 mmol) in 1,4-dioxane/H2O mixture (5:1, products (6a-6h) were purified by column chromatography.
20
mL),
followed
by
Na2CO3
(3.32
g,
31.62
mmol),
6-(4-(methylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (6a). Yellow solid: yield 53.0%, mp 301.1-302.0 °C.
1H NMR (400 MHz, DMSO-d6): δ = 9.49 (s, 1H), 9.16 (s, 1H), 8.65 (d, J = 9.0
Hz, 1H), 8.19 (d, J = 8.1 Hz, 2H), 8.13 (d, J = 7.6 Hz, 4H), 8.07 (d, J = 9.1 Hz,
1H), 7.67 (s, 3H), 3.32 (s, 3H). ESI-QTOF-MS: 445.0891 (C23H17N4O4S, M+H+),
Anal. Calcd for C23H16N4O4S: 444.0892.
Dichlorobis(triphenylphosphine)palladium(II) (220 mg, 0.32 mmol). The
mixture was then heated and stirred at 60 °C under nitrogen atmosphere for
8 h. After being cooled to room temperature, the mixture was diluted with
DCM (120 mL), washed with H2O (3×40 mL), and dried (Na2SO4). The solvent
was then evaporated under reduced pressure, and the residue was purified
by chromatography to afford white solid 3 (3.7 g). Yield: 94.1%, mp, 233.5-
1
234.5 °C. H NMR (400 MHz, CDCl3) δ = 9.52 (s, 1H), 9.08 (s, 1H), 8.21 (d, J =
6-(4-(methylsulfonyl)phenyl)-3-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)-1,8-
9.0 Hz, 1H), 8.13 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 8.3 Hz,
2H), 4.44 (q, J = 7.1 Hz, 2H), 3.13 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). ESI-QTOF- 1H NMR(400 MHz, DMSO-d6) δ = 9.49 (s, 1H), 9.30 (s, 1H), 9.19 (s, 1H), 8.84
naphthyridin-4(1H)-one (6b). Yellow solid: yield 49.1%, mp 297.7-299.2 °C.
MS: 395.0766 (C23H16N4O4SNa, M+Na+), Anal. Calcd for C18H16N2O5S:
(s, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.49 (d, J = 6.8 Hz, 1H), 8.16 (d, J = 14.9 Hz,
4H), 8.07 (d, J = 8.1 Hz, 1H), 7.70 (s, 1H). 3.32 (s, 3H). ESI-QTOF-MS:
446.0847 (C22H16N5O4S M+H+), Anal. Calcd for C22H15N5O4S, 445.0845.
372.0780.
Synthesis
of
6-(4-(methylsulfonyl)phenyl)-4-oxo-1,4-dihydro-1,8-
6-(4-(methylsulfonyl)phenyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (6c). Yellow solid: yield 60.3%, mp 320.9-321.6 °C.
1H NMR (400 MHz, DMSO-d6) δ = 9.51 (s, 1H), 9.20 (s, 1H), 8.89 (d, J = 5.3 Hz,
2H), 8.67 (d, J = 9.5 Hz, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.3 Hz, 2H),
8.09 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 5.4 Hz, 2H), 3.32 (s, 3H). ESI-QTOF-MS:
446.0848 (C22H16N5O4S, M+H+), Anal. Calcd for C22H15N5O4S, 445.0845.
naphthyridine-3-carbohydrazide (4)
3 (2.68 mmol) obtained by a known method was suspended in methanol
(15 mL) and to this mixture, hydrazine monohydrate (1 mL) was added
dropwise at room temperature for 3 h. The precipitate was separated from
the reaction mixture by means of suction filtration to give the 4 (600 mg).
Yield: 62.3%.1H NMR (400 MHz, DMSO-d6) δ = 9.77 (s, 1H), 9.42 (s, 1H), 9.05
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 9 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
yellow solid (250 mg, yield 65.9%). 1H NMR (400 MHz, DMSO-d6): δ = 9.27 (d,
J = 2.2 Hz, 1H), 9.14 (s, 1H), 8.35 (dd, J = 9.3 Hz, J = 2.2 Hz, 1H), 8.13 (d, J =
8.6 Hz, 2H), 7.88 (d, J = 9.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H).
6-(4-(methylsulfonyl)phenyl)-3-(5-(pyridin-2-yl)-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (6d). Yellow solid: yield 42.2%, mp 291.2-292.3 °C.
1H NMR (400 MHz, DMSO-d6): δ = 9.48 (d, J = 2.0 Hz, 1H), 9.11 (s, 1H), 8.83
(d, J = 4.8 Hz, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.64 (d, J = 2.2 Hz, 1H), 8.27 (d, J =
7.8 Hz, 1H), 8.18 (d, J = 8.5 Hz, 4H), 8.09 (dd, J = 9.5 Hz, J = 5.3 Hz, 2H), 7.67
(dd, J = 7.5 Hz, J = 5.0 Hz, 1H). 3.32 (s, 3H). ESI-QTOF-MS: 446.0847
(C22H16N5O4S, M+H+), Anal. Calcd for C22H15N5O4S, 445.0845.
Synthesis of 6-bromo-3-(5-(4-chlorophenyl)-1, 3, 4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (12)
Using a method similar to that of 9a, compound 9b was synthesized as a
yellow solid (280 mg, yield 40.8%). 1H NMR (400 MHz, DMSO-d6): δ = 9.26 (d,
J = 1.8 Hz, 1H), 9.12 (s, 1H), 8.34 (dd, J = 9.3 Hz, J = 2.2 Hz, 1H), 8.17 (dd, J =
8.8 Hz, J = 5.4 Hz, 2H), 7.87 (d, J = 9.3 Hz, 1H), 7.50 (t, J = 8.9 Hz, 2H).
6-(4-(methylsulfonyl)phenyl)-3-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (6e). Yellow solid: yield 47.4%, mp 311.2-312.9 °C.
1H NMR (400 MHz, DMSO-d6): δ = 9.49 (d, J = 1.9 Hz, 1H), 9.15 (s, 1H), 8.64
(dd, J = 9.1 Hz, J = 2.1 Hz, 1H), 8.19 (d, J = 8.5 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H),
8.06 (d, J = 9.1 Hz, 1H), 7.94 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.6 Hz,
1H), 7.48 (d, J = 7.5 Hz, 1H), 3.32 (s, 3H), 2.45 (s, 3H). ESI-QTOF-MS:
459.1051 (C24H19N4O4S, M+H+), Anal. Calcd for C24H18N4O4S, 458.1049.
General procedure for the synthesis of compounds 10a-10c and 13a-13c
Appropriate boronic acid or boronic acid ester (0.089 mmol) was added to
a solution of intermediate 9a-9b (0.074 mmol) in 1,4-dioxane/H2O mixture
(5:1,
5
mL), followed by Na2CO3 (23 g, 0.223 mmol),
3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-(4-(methylsulfonyl)phenyl)-
1,8-naphthyridin-4(1H)-one (6f). Yellow solid: yield 55.0%, mp 320.0-320.9
°C. 1H NMR (400 MHz, DMSO-d6): δ = 9.49 (s, 1H), 9.16 (s, 1H), 8.65 (dd, J =
9.1 Hz, J = 1.9 Hz, 1H), 8.18 (d, J = 8.3 Hz, 2H), 8.13 (d, J = 7.6 Hz, 4H), 8.07 (d,
dichlorobis(triphenylphosphine)palladium(II) (3 mg, 0.004 mmol). The
mixture was then heated and stirred at 60 °C under nitrogen atmosphere for
8 h. After being cooled to room temperature, the mixture was diluted with
J = 9.1 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 3.31 (s, 3H). ESI-QTOF-MS: 479.0505 DCM, washed with H2O, and dried (Na2SO4). The solvent was then
(C23H16ClN4O4S, M+H+), Anal. Calcd for C23H15ClN4O4S, 478.0503.
evaporated under reduced pressure, and the residue was purified by
chromatography to afford compounds 10a-10c. Compounds 13a-13c were
3-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(4-(methylsulfonyl)phenyl)-
synthesized by the similar method.
1,8-naphthyridin-4(1H)-one (6g). Yellow solid: yield 50.4%, mp 307.3-308.2
°C. 1H NMR (400 MHz, DMSO-d6): δ = 9.49 (d, J = 2.0 Hz, 1H), 9.16 (s, 1H),
3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-4-oxo-6-(pyridin-3-yl)-1,4-
8.65 (dd, J = 9.1 Hz, J = 2.2 Hz, 1H), 8.21 – 8.19 (m, 1H), 8.18 (d, J = 2.2 Hz,
2H), 8.13 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 9.1 Hz, 1H), 7.51 (t, J = 8.9 Hz, 2H),
3.32 (s, 3H). ESI-QTOF-MS: 463.0800 (C23H16FN4O4S, M+H+), Anal. Calcd for
dihydro-1,8-naphthyridin-1-ium (10a). Yellow powder: yield 80.4%, mp
305.5-306.5 °C. 1H NMR (400 MHz, DMSO-d6): δ = 9.47 (s, 1H), 9.16 (s, 1H),
9.10 (s, 1H), 8.73 (d, J = 4.8 Hz, 1H), 8.65 (d, J = 8.9 Hz, 1H), 8.33 (d, J = 8.0
Hz, 1H), 8.13 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 9.4 Hz, 1H), 7.74 (d, J = 8.5 Hz,
2H), 7.67–7.56 (m, 2H). ESI-QTOF-MS: 402.0682 (C21H13ClN5O2 M+H+), Anal.
Calcd for C21H12ClN5O2, 401.0680.
C
23H15FN4O4S, 462.0798.
6-(4-(methylsulfonyl)phenyl)-3-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (6h). Yellow solid: yield 59.2%, mp 300.0-301.2 °C.
1H NMR (400 MHz, DMSO-d6): δ = 9.48 (d, J = 1.7 Hz, 1H), 9.13 (s, 1H), 8.64
(dd, J = 9.1 Hz, J = 2.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H),
8.05 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 3.32
(s, 3H), 2.42 (s, 3H). ESI-QTOF-MS: 459.1051 (C24H19N4O4S M+H+), Anal. Calcd
for C24H18N4O4S, 458.1049.
4-(6-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-oxo-5,8-dihydro-1,8-
naphthyridin-3-yl)benzonitrile (10b). Yellow powder: yield 80.1%, mp
292.5-293.5 °C. 1H NMR (400 MHz, DMSO-d6): δ = 9.48 (s, 1H), 9.16 (s, 1H),
8.64 (d, J = 8.9 Hz, 1H), 8.16–8.10 (m, 2H), 8.08 (s, 1H), 8.07-8.04 (m, 1H),
7.74 (d, J = 8.4 Hz, 2H). ESI-QTOF-MS: 426.0683 (C23H13ClN5O2, M+H+), Anal.
Calcd for C23H12ClN5O2, 425.0680.
Synthesis
of
6-bromo-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-phenyl-1,8-naphthyridin-
4(1H)-one (10c). Yellow powder: yield 81.3%, mp 238.9-239.2 °C. 1H NMR
(400 MHz, DMSO-d6): δ = 9.39 (d, J = 1.9 Hz, 1H), 9.13 (s, 1H), 8.60 (dd, J =
9.1 Hz, J = 2.1 Hz, 1H), 8.12 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 9.1 Hz, 1H), 7.88 (t,
J = 8.0 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.61 (t, J = 7.4 Hz, 2H), 7.53 (t, J = 7.3
Hz, 1H). ESI-QTOF-MS: 401.0730 (C22H14ClN4O2, M+H+), Anal. Calcd for
C22H13ClN4O2, 400.0727.
carbohydrazide (7)
Using a method similar to that of 4, compound 7 was synthesized as a
pink solid 7 (340 mg), Yield: 23.6%. 1H NMR (400 MHz, DMSO-d6): δ = 9.73 (s,
1H), 9.23 (d, J = 1.8 Hz, 1H), 9.02 (s, 1H), 8.32 (dd, J = 9.3 Hz, J = 2.1 Hz, 1H),
7.84 (d, J = 9.3 Hz, 1H), 4.79 (s, 2H).
Synthesis of compounds 8 and 11
3-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(pyridin-3-yl)-1,8-
Using a method similar to that of 5a, compound 8 and 11 was synthesized
naphthyridin-4(1H)-one (13a). Yellow powder: yield 82.4%, mp 306.2-307.1
°C. 1H NMR (400 MHz, DMSO-d6): δ = 9.47 (s, 1H), 9.15 (s, 1H), 9.10 (s, 1H),
8.73 (s, 1H), 8.65 (d, J = 8.9 Hz, 1H), 8.33 (d, J = 7.3 Hz, 1H), 8.19 (s, 1H), 8.06
(d, J = 9.1 Hz, 1H), 7.62 (s, 1H), 7.51 (t, J = 8.6 Hz, 2H). ESI-QTOF-MS:
386.0978 (C21H13FN5O2, M+H+), Anal. Calcd for C21H12FN5O2, 385.0975.
as a brown solid. Yield: 76.1% and 90.0%, respectively.
Synthesis
of
6-bromo-3-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one (9)
To a mixture of 8 (0.94 mmol) in SOCl2 (5 mL), pyridine (160 mg, 2 mmol)
was added dropwise. The reaction mixture was then gradually heated to the 3-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(pyridin-4-yl)-1,8-
naphthyridin-4(1H)-one (13b). Yellow powder: yield 76.2%, mp 260.1-262.1
°C. 1H NMR (400 MHz, DMSO-d6): δ = 9.53 (d, J = 1.8 Hz, 1H), 9.15 (s, 1H),
8.78 (d, J = 5.1 Hz, 2H), 8.65 (dd, J = 9.1 Hz, 2.0, 1H), 8.18 (dd, J = 8.7 Hz, J =
5.4 Hz, 2H), 8.06 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 5.8 Hz, 2H), 7.50 (t, J = 8.8 Hz,
2H). ESI-QTOF-MS: 386.0978 (C21H13FN5O2, M+H+), Anal. Calcd for
C21H12FN5O2, 385.0975.
reflux temperature and maintained there for 3 h. The remaining SOCl2 was
removed by rotary evaporation and poured into saturated NaHCO3 solution
to acidify to pH 7. Then the aqueous layer was extracted with CH2Cl2, and
the combined organic layers were dried over Na2SO4, filtered, and
concentrated. The product 9a was purified by column chromatography as a
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 10 of 11
View Article Online
DOI: 10.1039/C8MD00464A
ARTICLE
Journal Name
4-(6-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-5-oxo-5,8-dihydro-1,8-
(Luminata Crescendo Western HRP Substrate or Immobilon Western
naphthyridin-3-yl)benzonitrile (13c). Yellow powder: yield 86.1%, mp 339.0- Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA).
340.5 °C. 1H NMR (400 MHz, DMSO-d6): δ = 9.48 (s, 1H), 9.16 (s, 1H), 9.03 (s, The membranes were probed for GAPDH to confirm equal loading. In this
1H), 8.63 (d, J = 8.9 Hz, 1H), 8.22-8.15 (m, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.08 study, the anti-CHK1 primary antibody (220064) was a product of Zen
(s, 1H), 8.05 (d, J = 8.9 Hz, 1H). ESI-QTOF-MS: 410.1047 (C21H13FN5O2, M+H+), Bioscience Co., Ltd. (Chengdu) and GAPDH primary antibody (AF5718) was
Anal. Calcd for C21H12FN5O2, 409.0975.
purchased from R&D Systems, Inc. (Minneapolis, MN).
Cell lines and culture
Molecular docking
A549, HepG2, KB, MCF-7, Hela, and HCT116 cancer cells were maintained
in Dulbecco’s minimum essential medium (DMEM, Gibco, USA) or Roswell
Park Memorial Institute-1640 (RPMI-1640, Gibco, USA), supplemented with
10% fetal bovine serum (FBS; Gibco, Auckland, N.Z.), 100 U/mL penicillin and
100 U/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37
°C. In addition, the human normal hepatocyte cell line LO2 was introduced
in this study to evaluated the cytotoxicity of 3-(1,3,4-oxadiazol-2-yl)-1,8-
naphthyridin-4(1H)-one derivatives.
Structure based docking studies were carried out by using GOLD V3.0.1.
3D conformations of the 2 molecules were generated and minimized using
molecular mechanics (MM2) method and hamiltonian approximations
Austin model 1 (AM1) method available in the MOPAC2009. The RESP
charges were assigned to the molecules by 3 steps: at first, ESP charges of
the molecule were calculated (HF/6-31G* OPT ESP) based on the structure
using Gaussian 03; then the restrained ESP fitting was conducted using
Antechamber program in the AMBER; at last, the obtained RESP charges
were assigned to the Sybyl mol2 files of each molecule. The kinase domain
of ATR was built by homology modelling with PI3K γ as template. A sphere of
10 Å around the binding site in the ATR kinase domain was defined as the
docking site. RMSD of early termination was set to 1.5 Å. GA parameters
was set to GOLD default and for each molecule, a number of 30 dockings
were performed.
Cell viability assay by MTT
Cells were seeded into 96-well plate 24
h before experimental
manipulation. Then, cells were treated with the compounds. Cells were
treated with compound alone for 24 h before combination with cisplatin
(2.5 μM) for 48 h. After incubation, 20 μL of
a 5 mg/mL 3-(4,5-
Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was
added, and the plates were incubated for an additional 2-4 h at 37 °C. The
medium was subsequently removed, and DMSO was then added. The OD570
was measured using a Spectra MAX M5 microplate spectrophotometer
(Molecular Devices, CA, USA). For combination treatment, cells were treated
with compounds alone for 24 h before combination with cisplatin (2.5 μM)
for 48 h.
References
1
2
3
4
5
6
7
M.H. Hsu, C.Y. Liu, C.M. Lin, Y.J. Chen, C.J. Chen, Y.F. Lin, L.J. Huang,
K.H. Lee, S.C. Toxicol Appl Pharmacol. 2011, 259, 219-226.
K. Chen, S.C. Kuo, M.C. Hsieh, A. Mauger, C.M. Lin, E. Hamel, K.H.
Lee. J Med Chem. 1997, 40, 2266-2275.
K.Chen, S.C. Kuo, M.C. Hsieh, A.Mauger, C.M. Lin, E. Hamel, K.H. Lee.
J.Med.Chem. 1997, 40, 3049-3056.
H.K. Gençer, S. Levent, U. Acar Çevik, Y. Özkay, S. Ilgın. Bioorg Med
Chem Lett. 2017, 27, 1162-1168.
C. M. Bébéar, O. Grau, A. Charron, H. Renaudin, D. Gruson, and C.
Bébéar. Antimicrob Agents Chemother. 2000, 44, 2719-2727.
T. Kuroda, F. Suzuki, T. Tamura, K. Ohmori, H. Hosoe. J Med Chem.
1992, 35, 1130-1136.
Morphological analysis by Hoechst staining
The morphological changes were evaluated by Hoechst 33258 staining.
HCT116 cells were treated with 6e/6h alone for 24 h before combination
with cisplatin (2.5 μM) for 48 h. Then, cells were fixed with 4%
paraformaldehyde and stained with a solution of Hoechst 33258 dye (5
μg/mL). Next, the nuclear morphology of cells was observed by fluorescence
microscopy (Zeiss, Axiovert 200, Germany), and the number of nuclei were
satisficed and analyzed.
V. Lucchesi, D.P. Hurst, D.M. Shore, S. Bertini, B.M. Ehrmann, M.
Allarà, L. Lawrence, A. Ligresti, F. Minutolo, G. Saccomanni, H. Sharir,
M. acchia, V. Di Marzo, M.E. Abood, P.H. Reggio, C. Manera. J Med
Chem. 2014, 57, 8777-8791.
Apoptosis analysis by FCM
8
9
G. Saccomanni, G.P. ascali, S.D. Carlo, D. Panetta, M. De Simone, S.
Bertini, S. Burchielli, M. Digiacomo, M. Macchia, C. Manera, P.A.
Salvadori. Bioorg Med Chem Lett. 2015, 25, 2532-2535.
A.M. Malfitano, C. Laezza, A. D’Alessandro, C. Procaccini, G.
Saccomanni, T. Tuccinardi, C. Manera, M. Macchia, G. Matarese, P.
Gazzerro, M. Bifulco. PLoS One. 2013, 8, e62511.
To detect the apoptosis effect of 6e/6h combined with cisplatin, the
Annexin V-FITC/PI apoptosis detection kit was used in this section. Briefly,
HCT116 cells were treated with 6e/6h alone for 24 h before combination
with cisplatin (2.5 μM) for 48 h. The cells were then harvested and washed
with cold PBS. After centrifugation, the cells were stained with Annexin V-
FITC and PI and analyzed by flow cytometry (Becton-Dickinson, USA).
10 C. Manera, V. Benetti, M.P. Castelli, T. Cavallini, S. Lazzarotti, F.
Pibiri, G. Saccomanni, T. Tuccinardi, A. Vannacci, A. Martinelli, P.L.
Ferrarini. J Med Chem. 2006, 49, 5947-5957.
11 N. Yadav, P. Kumar, A. Chhikara, M. Chopra. Biomed Pharmacother.
2017, 95, 721-730.
12 A. Andreani, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, M.
Rambaldi, Eur J Med Chem. 2001, 36, 743–746.
13 C.D. Morris, A. Rose, J. Curwen, A.M. Hughes, D.J. Wilson, D.J. Webb.
Br J Cancer. 2005, 92, 2148-2152.
Western blot analysis
HCT116 cells were collected by scraping and lysis in RIPA buffer (50 mM
Tris HCL, 1% NP-40, 0.1% SDS, 150 nM NaCl, 1 mM EDTA and 0.5% sodium
deoxycholate) containing protease and phosphatase inhibitors. The total
protein concentrations were measured by the enhanced BCA Protein Assay
Kit (Beyotime® Biotechnology, China). Equal amounts of protein were loaded
onto 12% SDS-PAGE gels (Willget Biotech, Shanghai, China) and were
electro-transferred to Millipore 0.2 μm PVDF membranes. After washing,
the membranes were blocked with 5% skimmed milk and incubated with the
primary antibody (anti-CHK1 mouse mAb) at 4 °C overnight. Antibodies were
detected with horseradish peroxidase (HRP)-conjugated secondary antibody
and developed with an enhanced chemiluminescence detection kit
14 J. S
ł
awi
ń
ski, K. Szafra
ń
K. Macur, M. Belka, T. B
ski, A. Pogorzelska, B. Żołnowska, A. Kawiak,
czek. Eur J Med Chem. 2017, 132, 236-248.
ą
15 F.A.F. Ragab, S.M. Abou-Seri, S.A. Abdel-Aziz, A.M. Alfayomy, M.
Aboelmagd. Eur J Med Chem. 2017, 138, 140-151.
16 M.D. Altıntop, B. Sever, G. Akalın Çiftçi, G. Turan-Zitouni, Z.A.
Kaplancıklı, A. Özdemir. Eur J Med Chem. 2018, 155, 905-924.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 11 of 11
View Article Online
DOI: 10.1039/C8MD00464A
Journal Name
ARTICLE
17 X. He, X.Y. Li, J.W. Liang, C. Cao, S. Li, T.J. Zhang, F.H. Meng. Bioorg 29 P. Borst, S. Rottenberg, J. Jonkers. Cell Cycle. 2008, 7, 1353-1359.
Med Chem Lett. 2018, 28, 847-852. 30 D.B. Longley, P.G. Johnston. J. Pathol. 2005, 205, 275-292.
18 J. Sun, S.Z. Ren, X.Y. Lu, J.J. Li, F.Q. Shen, C. Xu, H.L. Zhu. Bioorg Med 31 J.W. Harper, S.J. Elledge. Mol Cell. 2007, 28, 739-745.
Chem. 2017, 25, 2593-2600. 32 B.B. Zhou, S.J. Elledge. Nature. 2000, 408, 433-439.
19 M.J. Ahsan, A. Choupra, R.K. Sharma, S.S. Jadav, P. Padmaja, M.Z. 33 K. Krüger, K. Geist, F. Stuhldreier, L. Schumacher, L. Blümel, M.
Hassan, A.B.S. Al-Tamimi, M.H. Geesi, M.A. Bakht. Anticancer Agents
Med Chem. 2018, 18, 121-128.
Remke, S. Wesselborg, B. Stork, N. Klöcker, S. Bormann, W.P. Roos,
S. Honnen, G. Fritz. Cancer Lett. 2018, 430, 34-46.
20 R. Trummer, W. Rangsimawong, W. Sajomsang, M. Kumpugdee- 34 L. Duan, R.Z. Perez, M. Hansen, S. Gitelis, C.G. Maki. Cancer Biol
Vollrath, P. Opanasopit, P. Tonglairoum. MedChemComm. 2018, 8, Ther. 2014, 15, 1600-1612.
22967–22973 L.I. Toledo, M. Murga, O. Fernandez-Capetillo. Mol 35 J. Herůdková, K. Paruch, P. Khirsariya, K. Souček, M. Krkoška, O.
.
Oncol. 2011, 5, 368-373.
21 J.M. Furgason, M. Bahassi el. Targeting DNA repair mechanisms in
cancer. Pharmacol There. 2013, 137, 298-308.
22 L.H. Einhorn. Proc Natl Acad Sci U S A. 2002, 99, 4592-4595.
23 G.J. Peters, C.L. van der Wilt, C.J. van Moorsel, J.R. Kroep, A.M.
Bergman, S.P. Ackland. Pharmacol Ther. 2000, 87, 227-253.
24 A. Wilmes, C. Bielow, C. Ranninger, P. Bellwon, L. Aschauer, A.
Limonciel, H. Chassaigne, T. Kristl, S. Aiche, C. G. Huber, C. Guillou, P.
Vondálová Blanářová, P. Sova, A. Kozubík, A. Hyršlová Vaculová.
Neoplasia. 2017, 19, 830-841.
36 T. Iwata, T. Uchino, A. Koyama, Y. Johmura, K. Koyama, T. Saito, S.
Ishiguro, T. Arikawa, S. Komatsu, M. Miyachi, T. Sano, M. Nakanishi,
M. Shimada. PLos One. 2017, 12-24.
37 A. Peralta-Sastre, C. Manguan-Garcia, A.de Luis, C. Belda-Iniesta, S.
,
Moreno, R. Perona, I. Sanchez-Perez. Int J Biochem Cell Biol. 2010
42, 318-328.
Hewitt, M. O. Leonard, W. Dekant, F. Bois, P. Jennings, Toxicol. In 38 L.M. Pouliot, Y.C. Chen, J. Bai, R. Guha, S.E. Martin, M.M. Gottesman,
Vitro
,
2015
,
30, 117
–
127.
25 S.P. Jackson, J. Bartek. Nature. 2009, 461, 1071-1078.
M.D. Hall. Cance Res. 2012, 72, 5945-5955.
39 X. Hou, C. Yang, L. Zhang, T. Hu, D. Sun, H. Cao, F. Yang, G. Guo, C.
Gong, X. Zhang, A. Tong, R. Li, Y. Zheng. Biomaterials. 2017, 124,
195-210.
26 V.G. Gorgoulis, L.V. Vassiliou, P. Karakaidos, P. Zacharatos, A.
Kotsinas, T. Liloglou, M. Venere, R.A. Jr. Ditullio, N.G. Kastrinakis, B.
Levy, D. Kletsas, A. Yoneta, M. Herlyn, C. Kittas, T.D. Halazonetis. 40 J. Tang, J.F. He, C.L. Yang, Y. Mao, T.T. Hu, L.J. Zhang, H. Cao, A.P.
Nature. 2005, 434, 907-913.
27 J. Bartkova, Z. Horejsí, K. Koed, A. Krämer, F. Tort, K. Zieger, P.
Tong, X.R. Song, G.G. He, G. Guo, Y.F. Luo, X.N. Zhang, Y.M. Xie, Y.
Zheng. J. Nanoparticle Res 2014, 16, 2682-2699.
.
Guldberg, M. Sehested, J.M. Nesland, C. Lukas, T. Ørntoft, J. Lukas, J. 41 B. Fang. J Med Chem. 2014, 57, 7859-7873.
Bartek. Nature. 2005, 434, 864-870.
42 Q. Liu, S. Guntuku, X.S. Cui, S. Matsuoka, D. Cortez, K. Tamai, G. Luo,
S. Carattini-Rivera, F. DeMayo, A. Bradley, L.A. Donehower, S.J.
28 T.G. Oliver, K.L. Mercer, L.C. Sayles, J.R. Burke, D. Mendus, K.S.
Lovejoy, M.H. Cheng, A. Subramanian, D. Mu, S. Powers, D. Crowley,
R.T. Bronson, C.A. Whittaker, A. Bhutkar, S.J. Lippard, T. Golub, J. 43 L. Hao, J.Y. Shi, L.F. Lu, F. Wu, M. Zhou, X.Y. Hou, W.J. Zhang, Z.Y.
Elledge. Genes Dev. 2000, 14, 1448-1459.
Thomale, T. Jacks, E.A. Sweet-Cordero. Genes Dev. 2010, 24, 837-
852.
Ding, R. Li. Med Chem Res. 2014, 23: 747-758.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins