Nitric oxide-donating and reactive oxygen species-responsive prochelators based on 8-hydroxyquinoline as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2021.113153

Metal ion chelators based on 8-hydroxyquinoline (8-HQ) have been widely explored for the treatment of many diseases. When aimed at being developed into potent anticancer agent, a largely unmet issue is how to avoid nonspecific chelation of metal ions by 8

Full text of DOI:10.1016/j.ejmech.2021.113153

European Journal of Medicinal Chemistry 212 (2021) 113153
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Nitric oxide-donating and reactive oxygen species-responsive
prochelators based on 8-hydroxyquinoline as anticancer agents
,
,
,
, *
Yuxia Zhang a 1, Jiaxin Yang b 1, Tingting Meng a, Yajuan Qin a, Tingyou Li a **, Junjie Fu b
,
,
***
Jian Yin b
a School of Pharmacy, Nanjing Medical University, Nanjing, 211166, PR China
b Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Metal ion chelators based on 8-hydroxyquinoline (8-HQ) have been widely explored for the treatment of
many diseases. When aimed at being developed into potent anticancer agent, a largely unmet issue is
how to avoid nonspecific chelation of metal ions by 8-HQ in normal cells or tissues. In the current work, a
two-step strategy was employed to both enhance the anticancer activity of 8-HQ and improve its cancer
cell specificity. Considering the well-known anticancer activity of nitric oxide (NO), NO donor furoxan
was first connected to 8-HQ to construct HQ-NO conjugates. These conjugates were screened for their
cytotoxicity, metal-binding ability, and NO-releasing efficiency. Selected conjugates were further
modified with a ROS-responsive moiety to afford prochelators. Among all the target compounds, prodrug
HQ-NO-11 was found to potently inhibit the proliferation of many cancer cells but not normal cells. The
abilities of metal chelation and NO generation by HQ-NO-11 were confirmed by various methods and
were demonstrated to be essential for the anticancer activity of HQ-NO-11. In vivo studies revealed that
HQ-NO-11 inhibited the growth of SW1990 xenograft to a larger extent than 8-HQ. Our results showcase
a general method for designing novel 8-HQ derivatives and shed light on obtaining more controllable
metal chelators.
Received 10 September 2020
Received in revised form
31 December 2020
Accepted 1 January 2021
Available online 5 January 2021
Keywords:
Metal chelator
8-Hydroxyquinoline
Nitric oxide donor
ROS-Responsive prodrug
Anticancer agent
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
derivatives with metal ions play essential roles in their biological
activities. For example, 8-HQ and clioquinol (5-chloro-7-iodo-8-
8-Hydroxyquinoline (8-HQ) has long been known as an
important scaffold in the field of medicinal chemistry. The phar-
macological applications of 8-HQ derivatives have been widely
explored, revealing a broad range of bioactivities including neuro-
protective, antibacterial, anti-HIV, antileishmanial, and anticancer
effects [1e5]. Importantly, the 8-HQ scaffold acts as a chelator of
hydroxyquinoline, 1, Scheme 1) exhibit moderate affinity for Cu2þ
and Zn2þ, and therefore are able to modulate metal homeostasis
and inhibit metal-induced amyloid
b (Ab) aggregation in Alz-
heimer’s disease (AD) [6]. Compound 2 was reported as a highly
active iron chelator with potential application in the symptomatic
and neuroprotective treatment of Parkinson’s disease (PD) [7].
Transition metals like copper and iron are essential for cell
growth and are particularly demanded by high-proliferating cancer
cells. Although sophisticated mechanisms are adopted by human
body to strictly regulate the levels of copper and iron, dysregulated
homeostasis of these metal ions can happen and is closely involved
in tumor occurrence and progress. For example, copper is known to
be an important factor for tumor growth, angiogenesis, and
metastasis. Increased intratumoral copper or altered systemic
copper distribution is found in many cancer patients [8e10].
Similarly, many cancer cells are characterized by increased uptake
and restricted elimination of iron, leading to disruption of iron
several transition metal ions, such as Cu2þ, Fe2þ, Fe3þ, and Zn2þ
The metal-binding ability of 8-HQ is attributed to the nitrogen atom
of the pyridine ring and the oxygen atom of the phenol, both of
which are potential protonation sites. The interactions of 8-HQ
.
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: l_tingyou@njmu.edu.cn (T. Li), jfu@jiangnan.edu.cn (J. Fu),
jianyin@jiangnan.edu.cn (J. Yin).
1
Y. Zhang and J. Yang contributed equally.
https://doi.org/10.1016/j.ejmech.2021.113153
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Scheme 1. Representative 8-HQ derivatives.
metabolism and tumor progress [11,12]. Against this background,
metal chelators such as 8-HQ are emerging as novel anticancer
agents and their anticancer mechanisms mainly include two as-
pects. First, transition metals such as copper and iron are key
components of several important enzymes involved in energy
metabolism and DNA synthesis. For example, cytochrome c oxidase,
NADH dehydrogenase-2, and tyrosinase require copper for their
functions, and ribonucleotide reductase needs iron as its co-factor.
These enzymes are particularly essential for cancer cells, and
depletion of copper or iron by 8-HQ derivatives therefore exert
anticancer effects [5,12]. Second, the interaction of 8-HQ with
copper or iron forms redox-active metal complexes, which partic-
ipate in redox cycling of copper or iron to generate cytotoxic
reactive oxygen species (ROS) to damage lipids, proteins, DNA, and
other biomolecules of cancer cells. The reaction between hydrogen
peroxide (H2O2) and iron ion to generate highly toxic hydroxyl
radicals (HO$) is well known as Fenton reaction, and copper ion can
lead to the production of HO$ in a similar manner [13]. Several 8-
HQ derivatives with confirmed metal-binding abilities have been
reported as anticancer candidates (Scheme 1), such as 8-HQ-
derived Mannich bases 3e5 [3,14], bis-8-HQ benzylamine chelator
6 [15], polyaminoquinoline compound 7 [16], and endoperoxide-8-
HQ conjugate 8 [17].
In our attempt to develop novel anticancer agents based on 8-
HQ, we believe that one of the major unmet issue in this field lies
in how to address the cancer specificity of metal chelators. As
mentioned above, elevated levels of metals like copper and iron are
found in many cancer cells. This lays the foundation for targeting
cancer cells with 8-HQ derivatives. However, transition metal ions
are key elements ubiquitously found in the whole human body, and
uncontrolled depletion of these ions by chelators in non-cancerous
sites could cause severe systematic toxic events [10,12]. Prodrug
design is undoubtedly a useful strategy to address this problem by
temporally shielding the chelating ability of 8-HQ until selectively
activated in cancer cells. For example, Oliveri et al. developed 8-HQ
glycoconjugate prodrugs 9 and 10, which were activated by the
corresponding glycosidase to release 8-HQ [18,19]. However, the
cancer specificity of these prodrugs was not investigated.
selectivity of 8-HQ derivatives and avoid non-specific metal bind-
ing in normal cells and tissues.
In addition, as a useful drug design strategy, we propose that
connecting another cytotoxic moiety to 8-HQ would further
enhance the anticancer activity of the conjugate. Based on our
previous experience, we choose nitric oxide (NO) donor as the
second cytotoxic component considering: 1) High levels of NO are
known to show anti-proliferative activity [23,24]; 2) NO further
reacts with superoxide (O2ꢀ) to generate ONOOꢀ, which could be
treated as a supplement for ROS-mediated prodrug activation [23];
and 3) The ability of NO donors to work synergistically with other
anticancer compounds have been well demonstrated [25,26].
Bearing the above ideas in mind, a two-step procedure was
employed to design and screen novel 8-HQ derivatives (Scheme 2).
First, NO donor phenylsulfonyl furoxan was conjugated to the C2
position of 8-HQ using different linkers, and the afforded conju-
gates were screened for their anticancer effects, metal-binding
ability, and NO-releasing amount. Second, selected conjugates
from the first step were subjected to prodrug modification by
connecting an arylboronic acid/ester moiety to the hydroxyl group
of 8-HQ through a self-immolative linker. The hydroxyl-modified 8-
HQ precursors were unable to bind to metal ions until activated by
high levels of ROS in cancer cells. The combined use of NO donor
conjugation and prodrug modification is expected to endow the
novel 8-HQ derivatives with both enhanced anticancer activity and
improved cancer cell selectivity, and this strategy has never been
reported before.
2. Results and discussion
2.1. Synthesis of NO donor-conjugated 8-HQ derivatives
A panel of eight NO-donating 8-HQ derivatives were synthe-
sized as deciphered in Scheme 3. Phenylsulfonyl furoxan was
chosen as the NO donor due to its clarified mechanism of NO
release and demonstrated anticancer activity [27e31]. 3,4-
Bis(phenylsulfonyl)furoxan was prepared in two steps from thio-
phenol as reported [32]. 2-Aminoethanol, 3-aminopropanol, 1-(2-
hydroxyethyl)piperazine, and 4-hydroxypiperidine were then
employed as linkers and individually reacted with 3,4-
bis(phenylsulfonyl)furoxan in basic condition to give 3-
phenylsulfonyl-4-substituited furoxans 11aed, respectively [32].
All the four linkers have been well-documented in references for
constructing furoxan-based anticancer drugs [32e38]. 2-Methyl-8-
hydroxyquinoline was employed as another starting material due
Increased levels of ROS were regarded as a hallmark of cancer
and provide a potential trigger for prodrug design. Among the
numerous ROS-activated prodrugs, arylboronic acids and esters
represent the most common and well-studied moieties response
mainly to H2O2, and to a less extent, to peroxynitrite (ONOOꢀ)
[20e22]. We envision that installing an arylboronic acid/ester
moiety on 8-HQ scaffold will help to enhance the cancer cell
2

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Scheme 2. Design of novel NO-donating prochelators based on 8-HQ.
Scheme 3. Synthetic route of HQ-NO-1~HQ-NO-8. Reagents and conditions: a) 15% NaOH, THF, 0e25 ꢁC; b) SeO2, pyridine, 90 ꢁC; c) 11aed, EDCI, HOBT, DIPEA, DMF, 25 ꢁC; d) SeO2,
dioxane/H2O, 105 ꢁC; e) ethyl 2-(diethoxyphosphoryl)acetate, NaH, toluene, 25 ꢁC; f) LiOH, THF, 0e25 ꢁC; g) 11aed, EDCI, DIPEA, HOBT, CH2Cl2, 25 ꢁC.
to the easy and versatile modification of the 2-methyl group. Thus,
SeO2-mediated oxidation of the methyl group in pyridine or 1,4-
dioxane/H2O gave 8-HQ-2-carboxylic acid 12 and 8-HQ-2-
carbaldehyde 13, respectively. The carboxylic group of 12 was
coupled with the terminal amine groups of 11aed mediated by 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), hydrox-
ybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA) to
give NO-donating 8-HQ derivatives (HQ-NO) HQ-NO-1~HQ-NO-4.
Wittig-Horner reaction of aldehyde 13 with ethyl 2-(diethox-
hydrogen). The structures of all new compounds were validated by
1H NMR, 13C NMR, and mass spectroscopy (Supporting
Information).
2.2. Screening of HQeNOe1~HQeNOe8
The cytotoxicity of conjugates HQ-NO-1~HQ-NO-8 toward five
cancer cell lines (human non-small cell lung cancer A549, human
cervical carcinoma HeLa, human hepatocellular carcinoma HepG2,
human breast adenocarcinoma MCF-7, and human pancreatic
cancer PANC-1) was evaluated using MTT assay at concentrations of
yphosphoryl)acetate afforded ethyl a,b-unsaturated ester 14, which
was then hydrolyzed in LiOH to give carboxylic acid 15. In a similar
way to 12, 15 was condensed with 11aed in the presence of EDCI,
HOBT, and DIPEA to generate conjugates HQ-NO-5~HQ-NO-8. It is
noticeable that HQ-NO-5 and HQ-NO-6 adopted an E-configuration
as confirmed by 1H NMR spectrum (J ¼ 12.9 and 15.8 Hz, respec-
tively for the alkene hydrogen). In contrast, the conjugation of 15
with 11c and 11d gave Z-isomers HQ-NO-7 and HQ-NO-8 as the
main products (J ¼ 8.0 and 8.1 Hz, respectively for the alkene
10 mM and 20 mM, and 8-HQ was employed as the control. As shown
in Fig. 1, Table S1, and Table S2, PANC-1 was the most sensitive cell
line toward HQ-NO treatment, followed by MCF-7, HepG2, and
A549, and the proliferation of HeLa cells were least affected by
compound treatment. Among the eight HQ-NO conjugates,
a,b-
unsaturated amides HQ-NO-5~HQ-NO-8 showed less anticancer
activity than HQ-NO-1~HQ-NO-4. HQ-NO-2 and HQ-NO-4 were
3

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
(PANC-1 and SW1990) for both HQ-NO-2 and HQ-NO-4. In addi-
tion, as a commonly used method to study the anticancer activity of
metal chelators [14,39], cells were treated with HQ-NO-2 and HQ-
NO-4 in the presence of 50
effects of different transition metals on the cytotoxicity of chelators.
It was found that while 50 M CuCl2 or FeCl3 alone did not affect
mM CuCl2 or FeCl3 to investigate the
m
cell proliferation, CuCl2 greatly reduced the IC50 values of HQ-NO-2
and HQ-NO-4 to low micromolar or even submicromolar ranges in
PANC-1 and SW1990 cells. In contrast, FeCl3 had no significant ef-
fects on the IC50 values and even slightly increased the IC50 values
in some cell lines. These results suggest that the anticancer activity
of HQ-NO-2 and HQ-NO-4 might be mainly mediated by the for-
mation of redox active metal complexes and by inhibiting iron-
dependent enzymes (such as ribonucleotide reductase). There-
fore, the supplement of Cu2þ further facilitates the formation of
copper complex and induces more toxic ROS, leading to decreased
IC50 values. On the other hand, the addition of Fe3þ benefits the
generation of iron complex but meanwhile compromises the
enzyme inhibitory activity of the compounds, resulting in no sig-
nificant changes in IC50 values.
The Cu2þ and Fe3þ binding abilities of HQ-NO conjugates were
examined using the calcein competition assay [16,17]. In this assay,
the fluorescence of calcein (10 mM) is initially quenched by equal
mole of Cu2þ or Fe3þ to form calcein-Cu2þ or calcein-Fe3þcomplex.
If HQ-NO chelators are able to competitively bind to metal ions and
release calcein, recovered fluorescence will be observed. The results
shown in Fig. 2a and b and Table S3 clearly confirmed that all the
HQ-NO conjugates were able to recover the fluorescence after
40 min in a dose-dependent manner. Particularly, HQ-NO-2 and
HQ-NO-4 led to the highest recovery rates in both Cu2þ and Fe3þ
pretreated groups, indicating stronger metal ion binding abilities of
these two compounds than other candidates. The NO-releasing
capacity of HQ-NO-1~HQ-NO-8 in PANC-1 and SW1990 cells
Fig. 1. Cytotoxicity of HQ-NO-1~HQ-NO-8 toward five human cancer cell lines at a
concentration of 10
mM (a) and 20 mM (b) after a 48-h incubation. The percentages of
cell death were determined using MTT assay and are shown as mean SD (n ¼ 3).
were assessed using the Griess assay after incubation with 10 mM of
HQ-NO for 8 h. Obviously, in both PANC-1 (Fig. 2c) and SW1990
(Fig. 2d) cells, HQ-NO-2 and HQ-NO-4 generated prominent levels
of NO among all the tested compounds. Collectively, in quite good
accordance with the cytotoxic results, both the metal ion binding
and NO-releasing abilities of HQ-NO-2 and HQ-NO-4 outperformed
other candidates in our experiments. Therefore, these two com-
pounds were selected for prodrug modification.
found to be the most cytotoxic ones. At a concentration of 20 mM,
HQ-NO-2 and HQ-NO-4 led to 75.2% and 82.3% death of PANC-
1 cells, respectively, significantly higher than the effects of 8-HQ
(48.0%). These preliminary results suggest that, in accordance
with our design, NO donor conjugation is able to enhance the
cytotoxicity of some conjugates.
The IC50 values of HQ-NO-2 and HQ-NO-4 in the above five cell
lines and three additional cell lines (human colon cancer HCT116,
human prostate cancer PC3, and human pancreatic cancer cell
SW1990) were further measured to more accurately evaluate their
anticancer potency (Table 1). Interestingly, the most potent anti-
cancer activity was observed in human pancreatic cancer cell lines
2.3. Synthesis of ROS-responsive HQ-NO prodrugs
Prodrugs HQ-NO-9~HQ-NO-12 were facilely synthesized by
refluxing HQ-NO-2 or HQ-NO-4 with 2-(4-(bromomethyl)phenyl)-
Table 1
IC50 values of HQ-NO-1~HQ-NO-8 in cancer cells.a.
Cell lines
IC50 (mM)
CuCl2 (50
m
M)
FeCl3 (50
mM)
HQeNOe2
HQeNOe4
8-HQ
HQeNOe2
HQeNOe4
8-HQ
HQeNOe2
HQeNOe4
8-HQ
A549
HeLa
13.78 (0.53)
28.65 (2.13)
17.89 (2.01)
13.42 (1.56)
7.76 (0.54)
14.56 (1.20)
11.69 (0.88)
7.80 (0.66)
33.9 (2.54)
14.56 (1.74)
15.41 (1.03)
9.28 (1.12)
11.22 (0.80)
10.42 (0.92)
31.25 (4.20)
>50
>50
4.68 (0.35)
9.56 (0.84)
6.32 (0.62)
3.23 (0.12)
1.04 (0.07)
5.20 (0.61)
5.63 (0.44)
2.16 (0.17)
9.77 (1.01)
4.05 (0.52)
3.07 (0.21)
1.54 (0.23)
3.29 (0.28)
5.02 (0.47)
6.58 (0.57)
35.65 (2.12)
32.10 (2.66)
21.06 (1.89)
7.56 (0.87)
7.25 (0.74)
14.36
17.88 (2.30)
33.23 (3.05)
22.36 (2.58)
18.23 (1.47)
14.32 (1.69)
20.08 (2.32)
17.05 (2.22)
12.36 (0.97)
37.9 (4.02)
19.88 (2.11)
17.02 (1.47)
15.10 (2.06)
17.22 (1.69)
14.58 (1.32)
32.63 (4.11)
>50
>50
HepG2
MCF-7
PANC-1
HCT116
PC3
>50
>50
24.52 (3.11)
21.09 (3.25)
32.01 (2.59)
32.56 (3.98)
26.30 (2.88)
>50
(1.50)
SW1990
9.24 (0.74)
6.59 (0.58)
36.25 (3.02)
1.56 (0.20)
0.87 (0.05)
10.23
12.36 (1.04)
10.56 (1.07)
>50
(0.98)
a
Cells were incubated with indicated compounds in the presence or absence of 50
m
M of CuCl2 or FeCl3 for 48 h, IC50 values were measured by MTT assays and are shown as
mean (SD) (n ¼ 3).
4

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Fig. 2. The metal ion-binding and NO-releasing abilities of HQ-NO-1~HQ-NO-8. The fluorescence of calcein (10
m
M) was quenched by equal mole of Cu2þ or Fe3þ, and HQ-NO
chelators were added and incubated for 40 min. The Cu2þ (a) and Fe3þ (b) binding abilities of the chelators were evaluated from the recovered percentages of fluorescence.
Mean SD (n ¼ 3). The NO released from 10
mM of HQ-NO conjugates in PANC-1 (c) and SW1990 (d) cells after an 8-h incubation was measured using the Griess assay. Mean SD
(n ¼ 3).
4,4,5,5-tetramethyl-1,3,2-dioxaborolane or (4-(bromomethyl)
phenyl)boronic acid in K2CO3 and acetone. Both phenylboronic acid
and phenylboronic acid pinacol ester are known to response to ROS
to yield the phenolic hydroxyl group, followed by a spontaneous
1,6-elimination to regenerate HQ-NO-2 or HQ-NO-4 (Scheme 4)
[20,40].
2.4. Cytotoxicity of HQ-NO-9~HQ-NO-12
The cytotoxicity of HQ-NO-9~HQ-NO-12 toward human
pancreatic cancer cells PANC-1 and SW1990 were determined and
compared with that of HQ-NO-2, HQ-NO-4, and 8-HQ. As sum-
marized in Table 2, prodrug modification resulted in slightly to
Scheme 4. Synthetic route of prodrugs HQ-NO-9~HQ-NO-12 and ROS-mediated activation of HQ-NO-9~HQ-NO-12. Reagents and conditions: a) 2-(4-(bromomethyl)phenyl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane or (4-(bromomethyl)phenyl)boronic acid, K2CO3, Me2CO, reflux.
5

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Table 2
IC50 values tested compounds in cancer and normal cells.a.
Compounds
IC50 (mM)
CuCl2 (50
PANC-1
m
M)
FeCl3 (50
PANC-1
mM)
PANC-1
SW1990
H6C7
RWPE2
SW1990
SW1990
HQeNOe2
HQeNOe4
HQeNOe9
HQeNOe10
HQeNOe11
HQeNOe12
8-HQ
7.54 (0.68)
9.05 (1.12)
17.05 (2.05)
13.24 (1.24)
10.45 (0.80)
16.56 (1.32)
27.32 (1.68)
9.36 (1.02)
6.78 (0.77)
15.67 (1.45)
12.07 (1.05)
8.83 (0.78)
12.92 (1.58)
32.10 (4.21)
9.05 (1.10)
13.25 (0.82)
40.41 (3.54)
34.25 (2.57)
>50
8.77 (0.52)
7.04 (0.47)
39.78 (2.58)
28.62 (2.43)
35.62 (3.02)
30.08 (2.70)
25.63 (1.97)
0.86 (0.05)
1.32 (0.08)
4.89 (0.52)
3.47 (0.44)
1.69 (0.25)
2.69 (0.30)
7.43 (0.64)
1.47 (0.22)
0.79 (0.05)
3.20 (0.28)
2.88 (0.15)
1.20 (0.07)
3.05 (0.35)
11.50 (1.04)
13.89 (1.25)
14.78 (1.03)
25.32 (1.57)
38.25 (3.20)
17.56 (1.88)
20.50 (1.70)
30.44 (2.26)
12.06 (1.63)
11.23 (0.97)
20.78 (1.45)
34.21 (2.58)
15.40 (1.69)
18.70 (2.32)
>50
>50
20.12 (1.87)
a
Cells were incubated with indicated compounds in the presence or absence of 50
m
M of CuCl2 or FeCl3 for 48 h, IC50 values were measured by MTT assays and are shown as
mean (SD) (n ¼ 3).
moderately increased IC50 values relative to HQ-NO-2 and HQ-NO-
4. Among the four prodrugs, HQ-NO-11 showed the highest cyto-
toxicity to PANC-1 and SW1990 cells with an IC50 value of 10.45 and
2.5. Stability and ROS-responsive behaviors of prodrug HQ-NO-11
HQ-NO-9~HQ-NO-12 were decently stable in the absence of
ROS (Tables S4eS7 and Fig. 3a). After 24 h, more than 80% of intact
HQ-NO-11 were detected using HPLC in PBS buffers (pH 5.0, 7.4,
and 10.0) and cell culture medium (DMEM with 10% fetal bovine
serum) at 37 ꢁC. The ROS-responsive behaviors of HQ-NO-11 were
evaluated by treating the prodrug with different ROS (10 eq.)
(Fig. 3b). It was found that during 1 h, H2O2 treatment led to almost
complete decomposition (97.5%) of prochelator HQ-NO-11 and
generation of the corresponding chelator HQ-NO-2 (Fig. 3bed).
Peroxynitrite ONOOꢀ resulted in 20.8% of decomposition. Other
8.83 mM, respectively. To examine the cancer cell selectivity of the
tested compounds, IC50 values in two non-cancerous cell lines
(human pancreatic duct epithelial cell H6C7 and human prostate
normal cell RWPE2) were also measured. To our gratification, all the
prodrugs exhibited higher IC50 values in normal cells compared
with HQ-NO-2 and HQ-NO-4, strongly supporting an improved
cancer cell selectivity attributed to prodrug modification. The IC50
values in the presence of Cu2þ or Fe3þ were further determined,
showing similar effects on cytotoxicity as described in section 2.2.
Considering the high toxicity of HQ-NO-11 in cancer cells as well its
excellent cancer cell selectivity, this prodrug was chosen for further
in-depth studies.
t
ROSs including HO$, BuO$, ClOꢀ, and NO caused very little acti-
vation of the prodrug (Fig. 3b). This is in line with the reported ROS-
responsive behaviors of arylboronic acids and esters [20,41]. It is
noticeable that glutathione (GSH), Cu2þ, and Fe3þ did not affect the
stability of HQ-NO-11 in our experiments (Fig. 3b).
Fig. 3. Stability and ROS-responsive behaviors of prodrug HQ-NO-11. (a) Stability of HQ-NO-11 in PBS buffers and cell culture medium at 37 ꢁC. (b) Decomposition rate of HQ-NO-11
in the presence of ROS, GSH, Cu2þ, and Fe3þ after 1 h as detected by HPLC. (c) HPLC traces of HQ-NO-11 in the presence of 10 eq. of H2O2. (d) Time-dependent decomposition of HQ-
NO-11 in the presence of 10 eq. of H2O2.
6

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European Journal of Medicinal Chemistry 212 (2021) 113153
2.6. Cu2þ chelating ability of HQ-NO-11
2.7. Intracellular NO release from HQ-NO-11
Because the cytotoxicity of HQ-NO-11 toward cancer cells was
particularly higher in the presence of additional Cu2þ (Table 2), we
focused our efforts on investigating the Cu2þ chelating ability of
HQ-NO-11. First, it was unambiguously found that Cu2þ increased
the cytotoxicity of HQ-NO-11 toward SW1990 cells in a dose-
dependent manner (Fig. 4a and Table S8). The UV spectrum of
The NO release of HQ-NO-11 in SW1990 cells was measured
using the NO-sensitive fluorophore probe DAF-FM DA and analyzed
by flow cytometry. As shown in Fig. 6a, after a 24 h incubation, HQ-
NO-11 generated NO in SW1990 cells in a dose-dependent manner.
Compound 16 (Fig. 7a) was synthesized as a negative control
bearing a furazan moiety instead of a furoxan moiety, and thus is
not a NO donor. As expected, 16 treatment did not lead to any
detectable fluorescence in SW1990 cells. Western blot analysis
further revealed that HQ-NO-11 treatment increased the level 3-
HQ-NO-11 (50 mM) in the presence of CuCl2 (50 mM) were then
monitored. In the absence of H2O2, the UV absorbance curve
remained unchanged during 40 min (Fig. 4b) because the prodrug
was unable to chelate Cu2þ. Upon addition of H2O2 (10 eq.), instant
change of UV spectrum was observed showing increasing peaks at
240 and 382 nm, as well as shrinking peak at 210 nm (Fig. 4c). Using
calcein competition assay, it was further demonstrated that in the
presence of H2O2, HQ-NO-11 was able to bind to Cu2þ in time- and
dose-dependent manners (Fig. 4d). Finally, a Job’s plot was con-
structed by varying the ratio of HQ-NO-11 to Cu2þ and measuring
the UV absorbance at 240 nm in the presence of H2O2 to calculate
the binding stoichiometry [16,17]. A linear intercept maximum of
0.64 was found (Fig. 4e), confirming a preferential 1:2 (Cu2þ: HQ-
NO-2) binding mode.
The redox-active complexes formed between chelators and
metals are known to generate HO$ through Fenton or Fenton-like
reaction. Fortunately, Li et al. recently reported the first sensitive
probe capable of imaging HO$ in living cells [42]. We therefore
investigated the HO$ generation in SW1990 cells from HQ-NO-11
using this elegantly-designed probe via confocal laser scanning
microscope (CLSM). Treating cells with CuCl2 led to weak fluores-
cence due to the autooxidation of Cu2þ to generate HO$ (Fig. 5b). In
the presence of HQ-NO-11, the fluorescence was significantly
enhanced (Fig. 5c). Pretreating cells with HO$ scavenger tempol
greatly decreased the intensity of fluorescence even in the presence
of Cu2þ and HQ-NO-11 (Fig. 5d). These results further supported the
Cu2þ-binding ability of HQ-NO-11 in cancer cells.
nitrotyrosine-containing cytochrome
c
(3-NT Cyt-c) in
SW1990 cells (Fig. 6b) due to NO induced ONOOꢀ generation [43],
further confirming the NO-releasing ability of HQ-NO-11.
2.8. Anticancer mechanisms of HQ-NO-11
Two negative controls 16 and 17 were synthesized (Fig. 7a). As
described above, 16 was a structural mimic of HQ-NO-11 but
without NO-releasing ability. Compound 17 is analogous to HQ-NO-
11 but was unable to response to ROS, and thus is not supposed to
show metal binding property. As expected, neither 16 nor 17
showed obvious cytotoxicity in SW1990 cells at a concentration of
10 or 20 mM (Fig. 7b). Furthermore, when cells were pretreated with
ROS quencher N-acetylcysteine (NAC) or NO scavenger carboxyl-
PTIO, the cytotoxicity of HQ-NO-11 was significantly diminished.
These results confirmed that NO and ROS-mediated prodrug acti-
vation were essential to the anticancer activity of HQ-NO-11.
Using flow cytometry, it was found that HQ-NO-11 had limited
effects on the cell cycle distribution of SW1990 cells (Fig. 8a). Only
at a concentration of 12 mM did HQ-NO-11 slightly increase the
percentage of cells in G1 phase (Fig. 8a, column 3). However, HQ-
NO-11 significantly induced cell apoptosis (Fig. 8b, columns 2 and
3), to a much greater than 8-HQ (Fig. 8b, column 4). Compounds 16
and 17 had much lower apoptosis-inducing ability than HQ-NO-11
Fig. 4. The interaction of HQ-NO-11 with Cu2þ. (a) Cu2þ increased the cytotoxicity of HQ-NO-11 toward SW1990 cells in a dose-dependent manner. Mean SD (n ¼ 3). (b) UV
spectrum of HQ-NO-11 (50 m mM) during 40 min without H2O2. (c) UV spectrum of HQ-NO-11 (50 m mM) during 40 min in the presence of 10 eq.
M) and Cu2þ (50 M) and Cu2þ (50
H2O2. (d) The fluorescence of calcein (10
m
M) quenched by Cu2þ (10
m
M) was recovered in the presence of HQ-NO-11 and H2O2 during 40 min. (e) Job’s plot of HQ-NO-11 at pH ¼ 7.4.
Solutions with different HQ-NO-11/Cu2þ ratios were prepared in the presence of H2O2, and OD240 values were recorded after 1 h.
7

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Fig. 5. CLSM of SW1990 cells under different conditions (lex ¼ 570 nm, lem ¼ 650 nm), scale bar 20
m
M. (a) Cells only. (b) Cells preincubated with 20
m
M probe, then with 50
mM
CuCl2 for 30 min. (c) Cells preincubated with 20
m
M probe, then with 50
m
M CuCl2 for 30 min, and finally with 10
mM HQ-NO-11 for 1 h. (d) Cells preincubated with 20 m
M probe and
1 mM tempol, then with 50
mM CuCl2 for 30 min, and finally with 10
m
M HQ-NO-11 for 1 h. (e) Mean intensity from the second rows of images aed. Mean SD (n ¼ 3).
Fig. 6. Intracellular NO release from HQeNOe11 in SW1990 cells. (a) SW1990 cells were incubated with indicated compound for 48 h, and NO release was measured using the NO-
sensitive fluorophore probe DAF-FM DA and analyzed by flow cytometry. Mean SD (n ¼ 3). **p < 0.01 vs blank, ##p < 0.01 vs HQ-NO-11 treatments. (b) Cells were treated with the
indicated compounds for 24 h, or pretreated with carboxyl-PTIO (50 mM) for 1 h, followed by treatment with HQ-NO-11 for 24 h. The levels of 3-NT Cyt-c were detected by Western
blot. Mean SD (n ¼ 3). **p < 0.01 vs blank, ##p < 0.01 vs HQ-NO-11 treatments.
(Fig. 8b, columns 5 and 6), and either NAC or carboxyl-PTIO pre-
treatment decreased the apoptosis cells (Fig. 8b, columns 7 and 8).
A similar tendency regarding cells with decreased membrane po-
tential of mitochondria (Djm) was observed (Fig. 8c). Western blot
analysis revealed that HQ-NO-11 increased the expression of
cleaved Caspase 3, cleaved Caspase 9, and Bax-2, but decreased the
level of Bcl-2 (Fig. 8d and e). All the above results supported the fact
that HQ-NO-11 inhibited the proliferation of SW1990 cells via a
mitochondrial-dependent apoptosis-inducing pathway, and NO-
donating moiety significantly increased the anticancer activity of
HQ-NO-11.
2.9. In vivo anticancer activity of HQ-NO-11
Finally, the in vivo anticancer activity of HQ-NO-11 was evalu-
ated in SW1990-bearing nude mice model. Mice were randomly
divided into four groups and intravenously injected every three
days with saline, 10 mg/kg HQ-NO-11, 20 mg/kg HQ-NO-11, and
20 mg/kg 8-HQ, respectively. As shown in Fig. 9, HQ-NO-11 or 8-HQ
treatment significantly inhibited the tumor growth during the
three-week experiment compared with the saline treatment. After
21 days, the tumor sizes of mice treated with saline, 20 mg/kg HQ-
NO-11, and 20 mg/kg 8-HQ reached 1.23 0.25, 0.47 0.09, and
8

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
Fig. 7. The roles of NO and ROS-mediated prodrug activation in the anticancer activity of HQ-NO-11. (a) Structure comparison of HQ-NO-11, 16, and 17. (b) SW1990 cells were
pretreated with or without NAC (20 mM) or carboxyl-PTIO (50 M) for 1 h, and then treated with indicated compounds for 48 h, percentage of cell death was determined using MTT
assay. Mean SD (n ¼ 3). **p < 0.01 vs sole 20 M HQ-NO-11 treatment.
m
m
Fig. 8. The effects of tested compounds on cell cycle distribution (a), apoptosis (b), and decreased membrane potential of mitochondria (c) of SW1990 cells. Column 1: blank;
Column 2: 8
mM HQ-NO-11; Column 3: 12 mM HQ-NO-11; Column 4: 12 mM 8-HQ; Column 5: 12 mM 16; Column 6: 12 mM 17; Column 7: NAC pretreatment and then 12 mM HQ-NO-
11; Column 8: Carboxyl-PTIO pretreatment and then 12
compounds on the expression of apoptosis-related proteins. Mean SD (n ¼ 3).
m
M HQ-NO-11. Mean SD (n ¼ 3). **p < 0.01 vs column 1. ##p < 0.01 vs column 3. (d,e) Western blot analysis of tested
Fig. 9. Inhibitory effects of HQ-NO-11 on the growth of SW1990 nude mice xenografts. Mice were randomly divided into four groups and intravenously injected every three days
with saline, 10 mg/kg HQ-NO-11, 20 mg/kg HQ-NO-11, and 20 mg/kg 8-HQ, respectively. Volume change of tumors were monitored every two days and finally measured after
sacrificing mice on day 21. Mean SD (n ¼ 8).
9

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
0.68 0.19 cm3, respectively. At the same dose of 20 mg/kg, the
tumor inhibition rate of HQ-NO-11 treatment was calculated as
61.8%, significantly higher than that of the 8-HQ treatment (44.7%).
substituited furoxans 11aed were prepared according to the re-
ported method [38]. 8-Hydroxyquinoline-2-carboxylic acid (12)
and 8-hydroxyquinoline-2-carbaldehyde (13) were obtained based
on previously reported protocols [44,45]. RP-HPLC was run on
Agilent technologies 1260 infinity with a C18 column (Shim-pack
3. Conclusions
PREP-ODS(H) KIT (5
m
m, 250 mm ꢂ 4.6 mm)) at room temperature
The dysregulation of transition metal ions has become a
commonly recognized hallmark of cancer cells. Metal chelators
such as 8-HQ exert anticancer activities by both exhausting the
elevated metal ions and by forming redox-active metal complexes.
In the current work, we made efforts to develop novel 8-HQ de-
rivatives with enhanced anticancer activity and improved cancer
cell specificity. Our two-step strategy including NO donor conju-
gation and ROS-responsive prodrug modification led to the dis-
covery of HQ-NO-11 that meets our expected criteria.
HQ-NO-11 is a prochelator that is deprived of metal-binding
ability before activated by high levels of ROS (mainly by H2O2) in
cancer cells. This is important to avoid systematic side effects
caused by non-specific metal chelation in normal cells or tissues. In
MTT assays, the cytotoxicity of HQ-NO-11 toward cancer cells
PANC-1 and SW1990 is much higher than that toward normal cells
H6C7 and RWPE2, indicating the excellent cancer cell specificity of
HQ-NO-11. The metal-binding ability, particularly the Cu2þ
chelating ability of HQ-NO-11 after ROS-mediated activation was
confirmed by Calcein competition assays and by intracellular
detection of HO$ generation. In vitro, additional Cu2þ was found to
significantly increase the anticancer activity of HQ-NO-11, further
demonstrating the essential role of Cu2þ binding in HQ-NO-11
induced cancer cell death. In addition, the incorporation of NO
donor contributes to the greatly enhanced anticancer activity of
HQ-NO-11 as compared with 8-HQ, considering the fact that NO
quencher carboxyl-PTIO significantly diminished the cytotoxicity of
HQ-NO-11. Encouragingly, the tumor growth inhibitory rate of HQ-
NO-11 is higher than that of 8-HQ when administrated at a same
dose and frequency in SW1990-bearing nude mice.
Despite the above positive findings, several issues require
further investigations. First, the cancer specificity and systematic
safety of HQ-NO-11 demands more in-depth studies. Second, a
more comprehensive evaluation of the interactions between other
metal ions (such as Fe2þ and Zn2þ) and HQ-NO-11 is necessary to
further clarify the mechanism of action of this prochelator,
although our results clearly indicate mitochondrial-dependent
apoptosis-inducing pathway as a key factor. The particularly high
toxicity of most HQ-NO conjugates toward human pancreatic can-
cer cells is a thought-provoking phenomenon.
To conclude, our work provides a proof-of-concept for con-
structing novel 8-HQ derivatives with the potential for targeted
cancer therapy. Considering the fact that metal chelation by 8-HQ
can also be utilized to treat other diseases like AD and PD, it is
conceivable that the strategies employed in this work will find wide
applications in the field of medicinal chemistry.
under 256 nm. Eluants consisted of (A) 0.1% TFA in H2O and (B)
methanol. Gradients were 0e5 min, 40%B; 5e15 min 40e80%B;
15e20 min 80%B. The flow rate was 1 mL/min.
4.2. Synthetic procedures
4.2.1. Ethyl (E)-3-(8-hydroxyquinolin-2-yl)acrylate (14)
Compound 13 (4.62 mmol) was dissolved in toluene (7 mL) in
the presence of molecular sieves. NaH (6.94 mmol) and ethyl 2-
(diethoxyphosphoryl)acetate (4.62 mmol) were then added under
dark at r.t. and reacted at for 2 h. Upon completion, the solution was
adjusted to pH 7 with 1 M HCl and extracted with ethyl acetate. The
organic layers were combined, dried with anhydrous Na2SO4, fil-
trated, concentrated, and purified by column chromatography
(petroleum ether: ethyl acetate ¼ 1:3) to obtain a yellow solid
(930 mg, 83.4%). 1H NMR (400 MHz, CDCl3) (a mixture of E/Z ¼ 85/
15, and only the signals of E isomer are assigned)
d 8.20 (d,
J ¼ 8.5 Hz, 1H, ArH), 7.87 (d, J ¼ 15.9 Hz, 1H, CH), 7.64 (d, J ¼ 8.5 Hz,
1H, ArH), 7.50 (dd, J ¼ 8.3, 7.6 Hz, 1H, ArH), 7.35 (dd, J ¼ 8.2, 1.2 Hz,
1H, ArH), 7.22 (dd, J ¼ 7.7, 1.2 Hz, 1H, ArH), 7.02 (d, J ¼ 15.9 Hz, 1H,
CH), 4.35 (q, J ¼ 7.1 Hz, 2H, CH2), 1.41 (t, J ¼ 7.1 Hz, 3H, CH3).
4.2.2. (E)-3-(8-Hydroxyquinolin-2-yl)acrylic acid (15)
Compound 14 (3.83 mmol) was dissolved in THF/H2O (v/v, 2: 1,
10 mL) and LiOH (15.31 mmol) was slowly added at 0 ꢁC. The re-
action mixture was allowed to warm to r.t. and stirred for 1 h. Upon
completion, the solution was adjusted to pH 7 with 1 M HCl and left
to stand for precipitation. The mixture was filtered, and the solid
was washed with H2O, collected, and dried to obtain a yellow solid
(700 mg, 85.0%). 1H NMR (400 MHz, DMSO‑d6) (a mixture of E/
Z ¼ 88/12, and only the signals of E isomer are assigned)
d 10.87 (s,
1H, COOH), 8.58 (d, J ¼ 8.5 Hz, 1H, ArH), 7.81 (d, J ¼ 8.5 Hz, 1H, ArH),
7.54 (t, J ¼ 7.9 Hz, 1H, ArH), 7.47 (dd, J ¼ 8.3, 1.4 Hz, 1H, ArH), 7.28
(dd, J ¼ 7.6, 1.4 Hz, 1H, ArH), 7.24 (d, J ¼ 13.1 Hz, 1H, CH), 6.27 (d,
J ¼ 13.1 Hz, 1H, CH).
4.2.3. General procedure for target compounds HQ-NO-1~HQ-NO-
8
Acid 12 or 15 (0.31 mmol) was dissolved in anhydrous CH2Cl2
(5 mL) or anhydrous DMF (5 mL). EDCI (0.47 mmol), HOBT
(0.031 mmol), and DIPEA (0.47 mmol) were added, and the mixture
was stirred at r.t. for 1 h. Compounds 11aed were then added and
reacted overnight. Upon completion, the solution was concentrated
and purified by column chromatography (petroleum ether: ethyl
acetate ¼ 3:2) to obtain pure products.
4. Material and methods
4.2.4. 4-(2-(8-Hydroxyquinoline-2-carboxamido)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-1)
Yellow solid, yield 47.7%.
4.1. General information
1H NMR (400 MHz, DMSO‑d6)
d 10.13 (s, 1H, OH), 9.89 (d,
All reagents and solvents were received from commercial sup-
pliers and used without further purification. When necessary, sol-
vents were dried using standard protocols. TLC was performed on
silica gel plates and visualized under UV light (254 nm). Flash
chromatography was performed using chromatography grade silica
gel (200e300 mesh) purchased from Qingdao Haiyang Chemical
Co., Ltd. 1H NMR and 13C NMR spectrum were recorded with a JEOL
400 MHz spectrometer at 300 K. Mass spectrum were obtained on
an Agilent Q-TOF 6540 spectrometer. 3-Phenylsulfonyl-4-
J ¼ 5.9 Hz, 1H, NH), 8.55 (dd, J ¼ 8.6, 1.5 Hz, 1H, ArH), 8.17 (d,
J ¼ 1.5 Hz, 1H, ArH), 8.03e7.95 (m, 2H, ArH), 7.71 (dd, J ¼ 7.6, 1.5 Hz,
1H, ArH), 7.63e7.58 (m, 1H, ArH), 7.53 (td, J ¼ 7.9, 7.1, 1.6 Hz, 3H,
ArH), 7.22 (dd, J ¼ 7.6, 1.6 Hz, 1H, ArH), 4.66 (t, J ¼ 5.4 Hz, 2H, OCH2),
3.88 (q, J ¼ 5.1 Hz, 2H, NHCH2).
13C NMR (100 MHz, DMSO‑d6)
d 164.70 (C]O), 159.35 (quino-
line), 154.05 (furoxan), 147.60 (quinoline), 138.32 (Ph), 137.68
(quinoline), 136.87 (quinoline), 136.41 (Ph), 130.26 (Ph ꢂ 2), 130.03
(quinoline), 128.76 (Ph ꢂ 2, quinoline), 119.29 (quinoline), 118.08
10

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
(quinoline), 112.08 (quinoline), 111.00 (furoxan), 70.18 (OCH2),
38.03 (NCH2).
2H, ArH), 7.81 (dd, J ¼ 8.1, 5.2 Hz, 2H, ArH), 7.73 (dd, J ¼ 12.9, 5.8 Hz,
3H, ArH ꢂ 2, CH), 7.47e7.38 (m, 2H, ArH, CH), 7.31 (d, J ¼ 15.7 Hz,
1H, CH), 7.13 (d, J ¼ 7.4 Hz, 1H, ArH), 4.55 (t, J ¼ 5.2 Hz, 2H, OCH2),
3.70 (q, J ¼ 5.5 H, 2H, NHCH2).
MS (ESI, m/z): 479.1 [M þ Na]þ.
4.2.5. 4-(3-(8-Hydroxyquinoline-2-carboxamido)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-2)
Yellow solid, yield 48.1%.
13C NMR (100 MHz, DMSO‑d6)
d 165.25 (C]O), 158.85 (furoxan),
153.32 (quinoline), 151.49 (quinoline), 138.79 (CH]CH), 138.37
(quinoline), 137.17 (quinoline), 136.89 (quinoline), 136.04 (Ph),
129.98 (Ph ꢂ 2), 128.39 (Ph ꢂ 2), 128.01 (quinoline), 127.02 (CH]
CH), 120.98 (quinoline), 117.63 (quinoline), 111.68 (quinoline),
110.54 (furoxan), 69.74 (OCH2), 37.69 (NCH2).
1H NMR (400 MHz, DMSO‑d6)
d 10.07 (s, 1H, OH), 9.72 (t,
J ¼ 6.2 Hz, 1H, NH), 8.49 (d, J ¼ 8.5 Hz, 1H, ArH), 8.15 (d, J ¼ 8.5 Hz,
1H, ArH), 8.07 (d, J ¼ 7.8 Hz, 2H, ArH), 7.90 (t, J ¼ 7.5 Hz, 1H, ArH),
7.76 (t, J ¼ 7.6 Hz, 2H, ArH), 7.56 (t, J ¼ 7.9 Hz, 1H, ArH), 7.47 (d,
J ¼ 8.2 Hz, 1H, ArH), 7.18 (d, J ¼ 7.6 Hz, 1H, ArH), 4.52 (t, J ¼ 6.2 Hz,
2H, OCH2), 3.58 (t, J ¼ 6.6 Hz, 2H, CH2), 2.15 (q, J ¼ 6.5 Hz, 2H,
NHCH2).
MS (ESI, m/z): 483.2 [M þ H]þ, 505.2 [M þ Na]þ.
4.2.9. (E)-4-(3-(3-(8-Hydroxyquinolin-2-yl)acrylamido)propoxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-6)
Yellow solid, yield 50.1%.
13C NMR (100 MHz, DMSO‑d6)
d 163.92 (C]O), 158.94 (quino-
line), 153.53 (furoxan), 147.38 (quinoline), 137.71 (Ph), 137.17
(quinoline), 136.36 (quinoline), 136.13 (Ph), 130.04 (Ph ꢂ 2), 129.45
(quinoline), 129.36 (Ph ꢂ 2), 128.38 (quinoline), 118.80 (quinoline),
117.53 (quinoline), 111.49 (quinoline), 110.52 (furoxan), 69.43
(OCH2), 35.36 (NCH2), 28.65 (OCH2CH2).
1H NMR (400 MHz, DMSO‑d6)
d 9.69 (s, 1H, OH), 8.42 (t,
J ¼ 6.1 Hz, 1H, NH), 8.33 (d, J ¼ 8.5 Hz, 1H, ArH), 8.06 (d, J ¼ 7.8 Hz,
2H, ArH), 7.89 (d, J ¼ 7.6 Hz, 1H, ArH), 7.77 (d, J ¼ 7.9 Hz, 3H, ArH),
7.65 (d, J ¼ 15.8 Hz, 1H, CH), 7.46e7.36 (m, 2H, ArH), 7.24 (d,
J ¼ 15.7 Hz, 1H, CH), 7.11 (d, J ¼ 7.6 Hz, 1H, ArH), 4.47 (t, J ¼ 6.3 Hz,
2H, OCH2), 3.42e3.37 (m, 2H, CH2), 2.00 (dd, J ¼ 12.8, 6.2 Hz, 2H,
NHCH2).
MS (ESI, m/z): 471.3 [M þ H]þ, 493.3 [M þ Na]þ.
4.2.6. 4-(2-(4-(8-Hydroxyquinoline-2-carbonyl)piperazin-1-yl)
ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-3)
Yellow solid, yield 45.4%.
13C NMR (100 MHz, DMSO‑d6)
d 164.93 (C]O), 158.93 (furoxan),
153.30 (quinoline), 151.66 (quinoline), 138.43 (CH]CH), 138.33
(Ph), 137.17 (quinoline), 136.85 (quinoline), 136.17 (Ph), 130.07
(Ph ꢂ 2), 128.41 (Ph ꢂ 2), 127.98 (quinoline), 127.42 (CH]CH),
120.89 (quinoline), 117.64 (quinoline), 111.66 (quinoline), 110.55
(furoxan), 69.25 (OCH2), 35.25 (NCH2), 28.25 (OCH2CH2).
MS (ESI, m/z): 497.2 [M þ H]þ, 519.2 [M þ Na]þ.
1H NMR (400 MHz, CDCl3)
d
8.28 (d, J ¼ 8.5 Hz, 1H, ArH), 8.05 (d,
J ¼ 7.9 Hz, 2H, ArH), 7.73 (dd, J ¼ 8.0, 4.7 Hz, 2H, ArH), 7.61 (t,
J ¼ 7.7 Hz, 2H, ArH), 7.53 (t, J ¼ 7.9 Hz, 1H, ArH), 7.38 (d, J ¼ 8.2 Hz,
1H, ArH), 7.23 (d, J ¼ 7.5 Hz, 1H, ArH), 4.58 (t, J ¼ 5.4 Hz, 2H, OCH2),
3.90 (t, J ¼ 5.1 Hz, 2H, NCH2), 3.62 (t, J ¼ 5.0 Hz, 2H, NCH2),2 0.94 (t,
J ¼ 5.4 Hz, 2H, CH2), 2.76 (t, J ¼ 5.2 Hz, 2H, NCH2), 2.64 (t, J ¼ 5.0 Hz,
2H, NCH2).
4.2.10. (Z)-4-(2-(4-(3-(8-Hydroxyquinolin-2-yl)acryloyl)piperazin-
1-yl)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-
7)
13C NMR (100 MHz, CDCl3)
d
167.30 (C]O), 169.07 (quinoline),
152.38 (furoxan), 151.36 (quinoline), 138.15 (Ph), 137.69 (quinoline),
136.56 (quinoline), 135.82 (Ph), 129.81 (Ph ꢂ 2), 129.15 (quinoline),
128.64 (Ph ꢂ 2), 128.55 (quinoline), 121.58 (quinoline), 118.11
(quinoline), 111.14 (quinoline), 110.65 (furoxan), 69.36 (OCH2),
56.20 (NCH2), 53.88 (piperazine), 53.19 (piperazine), 47.44 (piper-
azine), 42.45 (piperazine).
Yellow solid, yield 40.3%.
1H NMR (400 MHz, DMSO‑d6)
d 9.69 (s, 1H, OH), 8.30 (d,
J ¼ 8.4 Hz, 1H, ArH), 8.03e7.98 (m, 3H, ArH), 7.84 (t, J ¼ 8.7 Hz, 2H,
ArH, CH), 7.71 (m, 3H, ArH), 7.40 (d, J ¼ 7.8 Hz, 1H, ArH), 7.35 (d,
J ¼ 8.0 Hz,1H, CH), 7.08 (d, J ¼ 7.4 Hz,1H, ArH), 4.51 (t, J ¼ 5.1 Hz, 2H,
OCH2), 3.75 (d, J ¼ 5.6 Hz, 2H, NCH2), 3.56 (t, J ¼ 5.0 Hz, 2H, NCH2),
2.79 (t, J ¼ 5.4 Hz, 2H, CH2), 2.60e2.48 (m, 2H, NCH2 ꢂ 2).
MS (ESI, m/z): 526.2 [M þ H]þ, 548.2 [M þ Na]þ.
13C NMR (100 MHz, DMSO‑d6)
d 164.10 (C]O), 158.89 (furoxan),
4.2.7. 4-((1-(8-Hydroxyquinoline-2-carbonyl)piperidin-4-yl)oxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-4)
Yellow solid, yield 39.7%.
153.21 (quinoline), 151.54 (quinoline), 140.77 (Ph), 138.07 (CH]
CH), 137.31 (Ph), 136.76 (Ph), 136.07 (quinoline), 130.01 (Ph ꢂ 2),
128.19 (Ph ꢂ 2), 123.49 (quinoline), 122.04 (CH]CH), 117.48
(quinoline), 114.21 (quinoline), 111.33 (quinoline), 110.46 (furoxan),
69.40 (OCH2), 55.40 (NCH2), 53.30 (piperazine), 52.52 (piperazine),
45.20 (piperazine), 41.77 (piperazine).
1H NMR (400 MHz, DMSO‑d6)
d 9.82 (s, 1H, OH), 8.41 (d,
J ¼ 8.5 Hz, 1H, ArH), 8.03e7.99 (m, 2H, ArH), 7.87 (t, J ¼ 7.5 Hz, 1H,
ArH), 7.73 (t, J ¼ 7.9 Hz, 2H, ArH), 7.65 (d, J ¼ 8.5 Hz, 1H, ArH),
7.50e7.45 (m, 1H, ArH), 7.41 (dd, J ¼ 8.2, 1.6 Hz, 1H, ArH), 7.13 (dd,
J ¼ 7.4, 1.4 Hz, 1H, ArH), 5.16 (tt, J ¼ 6.8, 3.5 Hz, 1H, CH), 3.83e3.71
(m, 2H, NCH2), 3.55e3.40 (m, 2H, NCH2), 2.05 (m, 2H, CH2),
1.94e1.74 (m, 2H, CH2).
MS (ESI, m/z): 552.3 [M þ H]þ, 574.2 [M þ Na]þ.
4.2.11. (Z)-4-((1-(3-(8-Hydroxyquinolin-2-yl)acryloyl)piperidin-4-
yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-8)
Yellow solid, yield 44.8%.
13C NMR (100 MHz, DMSO‑d6)
d 166.87 (C]O), 157.87 (quino-
line), 153.53 (furoxan), 151.97 (quinoline), 137.36 (Ph), 137.23
(quinoline), 136.93 (quinoline), 136.17 (Ph), 130.08 (Ph ꢂ 2), 128.64
(quinoline), 128.42 (Ph ꢂ 2, quinoline), 120.47 (quinoline), 117.80
(quinoline), 112.38 (quinoline), 110.53 (furoxan), 77.32 (OCH), 43.03
(piperidine), 37.90 (piperidine), 29.79 (piperidine), 29.23
(piperidine).
1H NMR (400 MHz, DMSO‑d6)
d 9.76 (s, 1H,OH), 8.35 (d,
J ¼ 8.7 Hz, 1H, ArH), 8.15e7.99 (m, 3H, ArH), 7.90 (t, J ¼ 7.7 Hz, 2H,
ArH, CH), 7.83e7.68 (m, 3H, ArH), 7.45 (t, J ¼ 7.8 Hz, 1H, ArH), 7.39
(d, J ¼ 8.1 Hz, 1H, CH), 7.12 (d, J ¼ 7.4 Hz, 1H, ArH), 5.18 (tt, J ¼ 6.8,
3.5 Hz, 1H, CH), 4.03e3.80 (m, 2H, NCH2), 3.68 (m, 2 H, NCH2), 2.03
(m, 2H, CH2),1.95e1.76 (m, 2H, CH2).
MS (ESI, m/z): 497.3 [M þ H]þ, 519.3 [M þ Na]þ.
13C NMR (100 MHz, DMSO‑d6)
d 164.33 (C]O), 157.87 (furoxan),
153.26 (quinoline), 151.57 (quinoline), 140.96 (CH]CH), 138.09
(Ph), 137.27 (quinoline), 136.83 (quinoline), 136.16 (Ph), 130.06
(Ph ꢂ 2), 128.42 (Ph ꢂ 2), 128.10 (Ph ꢂ 2), 123.58 (CH]CH), 122.09
(quinoline), 117.55 (quinoline), 111.40 (quinoline), 110.51 (furoxan),
77.33 (OCH), 41.56 (piperidine), 38.18 (piperidine), 30.53 (piperi-
dine), 29.36 (piperidine).
4.2.8. (E)-4-(2-(3-(8-Hydroxyquinolin-2-yl)acrylamido)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-5)
Yellow solid, yield 45.3%.
1H NMR (400 MHz, DMSO‑d6)
d
9.73 (s, 1H, OH), 8.53 (t,
J ¼ 5.5 Hz, 1H, NH), 8.35 (d, J ¼ 8.5 Hz, 1H, ArH), 8.05 (d, J ¼ 8.1 Hz,
11

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
MS (ESI, m/z): 523.2 [M þ H]þ, 545.2 [M þ Na]þ.
(quinoline), 154.62 (furoxan), 149.49 (quinoline), 139.51 (Ph),
138.81 (quinoline), 138.27 (Ph), 137.69 (quinoline), 136.66 (Ph),
134.80 (Ph ꢂ 2), 130.56 (Ph ꢂ 2), 128.99 (Ph ꢂ 2), 126.56 (Ph ꢂ 2),
120.46 (quinoline), 119.67 (quinoline), 112.25 (quinoline), 111.05
(Ph), 70.60 (OCH2Ph), 69.88 (OCH2), 36.16 (NCH2), 29.03
(CH2CH2CH2).
4.2.12. General procedure for target compounds HQ-NO-9~HQ-NO-
12
Compound HQ-NO-2 or HQ-NO-4 (0.2 mmol) was dissolved in
anhydrous acetone (10 mL). 2-(4-(Bromomethyl)phenyl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane or (4-(bromomethyl)phenyl)
boronic acid (0.22 mmol) and K2CO3 (0.2 mmol) were added, and
the reaction mixture was refluxed and monitored by TLC. Upon
completion, the solution was concentrated and purified by column
chromatography (petroleum ether: ethyl acetate ¼ 1:1).
MS (ESI, m/z): 627.4 [M þ Na]þ.
4.2.16. 4-((1-(8-((4-Boronobenzyl)oxy)quinoline-2-carbonyl)
piperidin-4-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
(HQ-NO-12)
White solid, yield 69.8%.
4.2.13. 3-(Phenylsulfonyl)-4-(3-(8-((4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)oxy)quinoline-2-carboxamido)propoxy)-
1,2,5-oxadiazole 2-oxide (HQ-NO-9)
1H NMR (400 MHz, DMSO‑d6)
d
8.46 (d, J ¼ 8.3 Hz, 1H, ArH),
8.03e7.98 (m, 3H, ArH), 7.86 (t, J ¼ 7.4 Hz, 1H, ArH), 7.80e7.77 (m,
2H, ArH), 7.75e7.70 (m, 2H, ArH), 7.59e7.53 (m, 2H, ArH), 7.49 (d,
J ¼ 7.7 Hz, 2H, ArH), 7.33 (dd, J ¼ 5.8, 3.3 Hz, 1H, ArH), 5.37 (s, 2H,
OCH2Ph), 5.17 (tt, J ¼ 7.2, 3.6 Hz, 1H, CH), 3.88 (dt, J ¼ 13.8, 5.0 Hz,
1H, NCH2), 3.61 (tq, J ¼ 9.2, 5.2, 4.5 Hz, 3H, NCH2), 2.10e1.78 (m, 4H,
NCH2 ꢂ 2).
White solid, yield 91.2%.
1H NMR (400 MHz, CDCl3)
d 8.51 (s, 1H,NH), 8.35e8.25 (m,2H,
ArH), 8.12e8.07 (m, 2H, ArH), 7.85 (d, J ¼ 7.7 Hz, 2H, ArH), 7.76e7.72
(m, 1H, ArH), 7.62 (t, J ¼ 7.8 Hz, 2H, ArH), 7.54 (d, J ¼ 7.7 Hz, 2H,
ArH), 7.47 (d, J ¼ 6.0 Hz, 2H, ArH), 7.12 (dd, J ¼ 6.0, 2.9 Hz, 1H, ArH),
5.43 (s, 2H, OCH2Ph), 4.55 (t, J ¼ 6.2 Hz, 2H, OCH2), 3.69 (q,
J ¼ 6.8 Hz, 2H, NCH2), 2.28e2.22 (m, 2H, CH2), 1.34 (s, 12H, CH3 ꢂ 4).
13C NMR (100 MHz, DMSO‑d6)
d 166.68 (C]O), 157.81 (quino-
line), 154.13 (furoxan), 152.38 (quinoline), 138.75 (Ph), 138.06
(quinoline), 137.24 (Ph), 136.10 (quinoline), 134.20 (Ph ꢂ 2), 130.03
(Ph ꢂ 2), 128.39 (Ph ꢂ 2), 127.93 (quinoline), 126.46 (Ph ꢂ 2), 120.91
(quinoline), 119.72 (quinoline), 111.03 (quinoline), 110.47 (furoxan),
77.18 (OCH), 69.92 (OCH2Ph), 42.95 (piperidine), 38.09 (piperidine),
30.09 (piperidine), 29.23 (piperidine).
13C NMR (100 MHz, CDCl3)
d 165.14 (C]O), 159.04 (quinoline),
154.65 (furoxan), 148.54 (quinoline), 140.02 (Ph), 138.89 (quino-
line), 138.17 (Ph),137.66 (quinoline),135.72 (Ph),135.24 (Ph),130.82
(Ph), 128.76 (Ph ꢂ 2), 128.41 (Ph ꢂ 2, quinoline), 128.28 (quinoline),
128.16 (quinoline), 126.37 (Ph), 120.25 (quinoline), 119.47 (quino-
line), 111.22 (quinoline), 110.59 (furoxan), 84.05 (CH ꢂ 2), 71.18
(OCH2Ph), 69.21 (OCH2), 36.10 (NCH2), 29.84 (CH2CH2CH2), 25.00
(CH3 ꢂ 4).
MS (ESI, m/z): 653.5 [M þ Na]þ.
4.2.17. (4-(((2-((3-((4-(Phenylsulfonyl)-1,2,5-oxadiazol-3-yl)oxy)
propyl)carbamoyl)quinolin-8-yl)oxy)methyl)phenyl)boronic acid
(16)
MS (ESI, m/z): 709.6 [M þ Na]þ.
HQ-NO-11 (0.2 mmol) was dissolved in trimethyl phosphite
(5 mL) and reacted under reflux for 10 h. After completion, the
solution was concentrated and purified by column chromatography
(petroleum ether: ethyl acetate ¼ 1:1).
4.2.14. 3-(Phenylsulfonyl)-4-((1-(8-((4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzyl)oxy)quinoline-2-carbonyl)piperidin-4-yl)
oxy)-1,2,5-oxadiazole 2-oxide (HQ-NO-10)
White solid, yield 88.6%.
White solid, yield 80.4%.
1H NMR (400 MHz, CDCl3)
d
8.25 (d, J ¼ 8.6 Hz, 1H, ArH), 8.04
1H NMR (400 MHz, CDCl3)
d
8.53 (d, J ¼ 8.3 Hz, 1H, ArH), 8.30 (d,
(m,2H, ArH), 7.88 (d, J ¼ 8.6 Hz, 2H,ArH), 7.81 (d, J ¼ 7.8 Hz, 2H,
ArH), 7.77e7.71 (m, 1H, ArH), 7.61 (t, J ¼ 7.9 Hz, 2H, ArH), 7.50 (d,
J ¼ 7.8 Hz, 2H, ArH), 7.47 (m, 2H, ArH), 7.10 (dd, J ¼ 7.5, 1.8 Hz, 1H,
ArH), 5.39 (s, 2H, OCH2Ph), 5.19 (tt, J ¼ 6.0, 3.4 Hz, 1H, CH),
4.16e3.74 (m, 4H, NCH2 ꢂ 2), 2.14 (dq, J ¼ 10.0, 5.3, 4.7 Hz, 2H,
NCH2), 1.98 (ddd, J ¼ 17.6, 9.3, 4.7 Hz, 2H, NCH2), 1.31 (s, 12H,
CH3 ꢂ 4).
J ¼ 8.5 Hz 2H, ArH), 8.21 (d, J ¼ 7.8 Hz, 2H, ArH), 8.01 (d, J ¼ 7.7 Hz,
2H, ArH), 7.65 (d, J ¼ 7.8 Hz, 3H, ArH), 7.56 (t, J ¼ 7.7 Hz, 2H, ArH),
7.48 (d, J ¼ 7.0 Hz, 2H, ArH), 7.18 (d, J ¼ 6.9 Hz, 1H, ArH), 5.46 (d,
J ¼ 7.1 Hz, 2H, OCH2Ph), 4.45 (t, J ¼ 6.4 Hz, 2H, OCH2), 3.59e3.50 (m,
2H, NCH2), 2.22e2.14 (m, 2H, CH2).
13C NMR (100 MHz, CDCl3)
d 165.09 (C]O), 161.21 (quinoline),
154.62 (furoxan),148.47 (quinoline),141.67 (Ph),138.85 (quinoline),
137.69 (Ph), 136.04 (quinoline), 135.55 (Ph), 130.52 (quinoline),
129.78 (Ph ꢂ 2), 129.04 (Ph ꢂ 2), 128.45 (Ph), 126.85 (Ph), 120.31
(quinoline), 119.43 (quinoline), 112.97 (quinoline), 111.12 (furoxan),
71.49 (OCH2Ph), 71.14 (OCH2), 36.01 (NCH2), 28.93 (CH2CH2CH2).
MS(ESI, m/z): 589.2 [M þ H]þ, 611.2 [M þ Na]þ.
13C NMR (100 MHz, CDCl3)
d 167.48 (C]O), 158.05 (quinoline),
154.70 (furoxan),151.96 (quinoline),139.90 (Ph),138.26 (quinoline),
137.82 (Ph), 137.26 (quinoline), 135.78 (Ph), 135.14 (Ph ꢂ 2), 129.85
(quinoline), 129.64 (quinoline), 128.58 (Ph ꢂ 2), 128.13 (quinoline),
126.56 (Ph ꢂ 2), 121.95 (quinoline), 119.92 (quinoline), 110.60
(quinoline), 84.00 (CH ꢂ 2), 75.15 (OCH), 70.79 (OCH2Ph), 43.78
(piperidine), 39.15 (piperidine), 30.88 (piperidine), 29.89 (piperi-
dine), 24.96 (CH3 ꢂ 4).
4.2.18. 4-(3-(8-(Benzyloxy)quinoline-2-carboxamido)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (17)
MS (ESI, m/z): 735.6 [M þ Na]þ.
HQ-NO-2 (0.12 mmol) was dissolved in ethanol (5 mL). Benzyl
bromide (0.15 mmol) and Na2CO3 (0.24 mmol) were added and the
mixture was allowed to react under dark overnight. After comple-
tion, the solution was concentrated and purified by column chro-
matography (petroleum ether: ethyl acetate ¼ 2:1).
White solid, yield 77.4%.
4.2.15. 4-(3-(8-((4-Boronobenzyl)oxy)quinoline-2-carboxamido)
propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (HQ-NO-11)
White solid, yield 71.3%.
1H NMR (400 MHz, DMSO‑d6)
d
8.71 (t, J ¼ 6.1 Hz, 1H, NH), 8.51
(d, J ¼ 8.6 Hz, 1H, ArH), 8.15 (d, J ¼ 8.5 Hz, 1H, ArH), 8.07e8.03 (m,
2H, ArH), 7.88 (t, J ¼ 7.5 Hz, 1H, ArH), 7.81 (d, J ¼ 7.9 Hz, 1H, ArH),
7.74 (t, J ¼ 7.8 Hz, 2H, ArH), 7.61e7.56 (m, 2H, ArH), 7.53 (d,
J ¼ 7.9 Hz, 2H, ArH), 7.31 (d, J ¼ 7.1 Hz, 2H, ArH), 5.47 (s, 2H,
OCH2Ph), 4.49 (t, J ¼ 6.0 Hz, 2H, OCH2), 3.54 (q, J ¼ 6.8 Hz, 2H,
NCH2), 2.11 (p, J ¼ 6.6 Hz, 2H, CH2).
1H NMR (400 MHz, CDCl3)
d
8.49 (t, J ¼ 6.3 Hz, 1H, NH),
8.34e8.27 (m, 2H, ArH), 8.12e8.05 (m, 2H, ArH), 7.74 (t, J ¼ 7.5 Hz,
1H, ArH), 7.62 (t, J ¼ 7.7 Hz, 2H, ArH), 7.55 (d, J ¼ 7.6 Hz, 2H, ArH),
7.52e7.44 (m, 2H, ArH), 7.40 (t, J ¼ 7.4 Hz, 1H, ArH), 7.33 (t,
J ¼ 7.3 Hz, 1H, ArH), 7.17 (dd, J ¼ 7.1, 1.6 Hz, 1H, ArH), 5.39 (s, 2H,
OCH2Ph), 4.53 (t, J ¼ 6.2 Hz, 2H, OCH2), 3.67 (q, J ¼ 6.7 Hz, 2H,
NCH2), 2.23 (q, J ¼ 6.5 Hz, 2H, CH2).
13C NMR (100 MHz, DMSO‑d6)
d 165.13 (C]O), 159.44
12

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
13C NMR (100 MHz, CDCl3)
d
165.12 (C]O), 159.03 (quinoline),
incubated with H2O2 (1 mM) at 37 ꢁC, and the reaction mixture was
monitored by HPLC.
154.82 (furoxan), 148.50 (quinoline), 138.94 (Ph), 138.20 (quino-
line), 137.63 (Ph), 136.92 (quinoline), 135.72 (Ph), 130.83 (quino-
line), 129.83 (Ph ꢂ 2), 128.73 (Ph ꢂ 2), 128.45 (Ph), 128.23 (Ph),
127.32 (Ph ꢂ 2), 120.20 (quinoline), 119.41 (quinoline), 111.15
(furoxan), 110.60 (quinoline), 71.28 (OCH2Ph), 69.16 (OCH2), 36.06
(NCH2), 29.05 (CH2CH2CH2).
4.8. UV spectrum measurement of HQ-NO-11
UV spectrum of HQ-NO-11 were recorded using a Shimadzu UV-
2600 spectrophotomer. Stock solution of HQ-NO-11 in DMSO was
diluted with PBS (10 mM, pH ¼ 7.4) buffer to make a final con-
MS (ESI, m/z): 583.3 [M þ Na]þ.
centration of 50 mM, followed by the addition of 50 mM CuCl2. The
4.3. MTT assays
UV spectrum were monitored at room temperature. In another
separate study, H2O2 (500 M) was added to the mixture of HQ-NO-
m
All the cell lines were purchased from American Tissue Culture
Collection (ATCC, Rockville, MD, USA). Cells were planked in a 96
well plate with a concentration of 6 ꢂ 104 cells per well and
cultured at 37 ꢁC with 5% CO2 for 24 h. Then cells were treated with
tested compounds and incubated for an additional 48 h. In case of
11 and CuCl2, and UV spectrum were monitored at room temper-
ature. To establish the Job’s plot, the ratio of [HQ-NO-11]/[CuCl2]
was varied from 0 to 1 while the total concentration ([HQ-NO-
11] þ [CuCl2]) was fixed to 100
mM H2O2 (500 mM) was added to the
mixture of HQ-NO-11 and CuCl2, and the UV the absorbance at
CuCl2 or FeCl3 co-incubation, 50
(5 mg/mL, 20
L) was added and the plate was kept at 37 ꢁC for
another 4 h. Then the medium was carefully removed. Dimethyl
sulfoxide (150 L per well) was added and oscillated gently to make
m
M CuCl2 or FeCl3 was added. MTT
240 nm was recorded after 1 h.
m
4.9. Intracellular HO· detection
m
crystal dissolved. The absorbance at 490 nm was measured using a
microplate reader. In case of NAC or carboxy-PTIO pretreatment,
The HO$ probe was synthesized according to the reported
method. When SW1990 cells grown in a 96-well plate reached 80%
confluence, they were washed with PBS. After being loaded with
cells were treated with 20 mM NAC or 50 mM carboxy-PTIO for
1 h at 37 ꢁC, and the following procedures were the same as
described above.
20
times with PBS, incubated with 50
or without 10 M HQ-NO-11 for 1 h. In the group of tempol control,
m
M HO$ probe at 37 ꢁC for 30 min, the cells were rinsed three
mM CuCl2 for 30 h, and then with
m
4.4. Calcein competition assay
cells pretreated with the probe were incubated with 1 mM tempol
for 30 min before adding CuCl2. CLSM images were recorded
The fluorescence emission (lex ¼ 485 nm, lem ¼ 520 nm) of
(
lex ¼ 570 nm, lem ¼ 650 nm) and mean intensity values were
calcein (10
m
M) in a 50 mM HEPES buffer was quenched by 10
mM
measured using ImageJ software.
CuCl2 or FeCl3. To screen the metal ion binding abilities of HQ-NO-
1~HQ-NO-8, the stock solution of individual chelator in DMSO was
added to the HEPES solution to reach a final concentration of 10 or
4.10. Intracellular NO detection by DAF-FM DA and flow cytometry
50
m
M. After incubation at room temperature for 40 min, the
The NO released from tested compounds in SW1990 cells were
measured using an NO-sensitive reagent fluorophore DAF-FM DA.
When cells grown in a 96-well plate reached 80% confluence, they
fluorescence was determined to calculate the recovery rate. To
study the copper binding ability of HQ-NO-11, the stock solution of
HQ-NO-11 in DMSO was added to the HEPES solution to reach a
final concentration of 10, 25, or 50
eq. of HQ-NO-11). The fluorescence was monitored over 40 min.
were washed with PBS. After being loaded with 5 mM DAF-FM DA at
mM, followed by adding H2O2 (10
37 ꢁC for 20 min, the cells were rinsed three times with PBS and
incubated with test compounds for 8 h. In case of carboxyl-PTIO
pretreatment, cells were treated with carboxyl-PTIO (50
mM) for
4.5. Intracellular Griess assay
1 h, and then incubated with 12 M HQ-NO-11 for 8 h. NO pro-
m
duction was measured with the flow cytometer (lex ¼ 495 nm,
SW1990 cells were treated with 10 mM of each compound for
lem ¼ 515 nm).
8 h. The nitrite content in each group was detected by the Griess
assay using the nitrite colorimetric assay kit (Beyotime, Nanjing,
China). The absorbance was recorded at 540 nm on a spectropho-
tometer (Smart spec, Bio-Rad) using NaNO2 as a standard to
establish the calibration curve.
4.11. 3-NT Cyt-c determination
SW1990 cells were treated with different indicated compounds
for 24 h, In case of NAC or carboxyl-PTIO pretreatment, cells were
treated with NAC (20 mM) or carboxyl-PTIO (50
then incubated with 12 M HQ-NO-11 for 24 h. Mitochondrial
proteins were extracted by centrifugation. After being lysed, the
mitochondrial protein lysates (30 g/lane, ~500 ng Cyt-c) were
separated by SDS-PAGE, transferred on PVDF membranes, and
probed with anti-3NT antibody (Proteintech Group Inc., IL, USA).
mM) for 1 h, and
4.6. Stability evaluation of HQ-NO-9~HQ-NO-12
m
A solution of HQ-NO-9~HQ-NO-12 (1 mM) in PBS (pH ¼ 5.0, 7.4,
m
or 10.0) with 5% DMSO or in cell culture medium (DMEM with 10%
fetal bovine serum) was stirred at 37 ꢁC. Aliquots (500
mL) of the
solution were analyzed by RP-HPLC to determine the remaining
concentrations of HQ-NO-9~HQ-NO-12.
4.12. Cell cycle analysis
4.7. ROS-behaviors of HQ-NO-11
SW1990 cells were planted at 5 ꢂ 105 per well in 6-well plate
and incubated for 24 h. Cells were treated with indicated com-
pounds for 24 h. In case of NAC or carboxyl-PTIO pretreatment, cells
Different ROS solutions were prepared according to previous
method. HQ-NO-11 (100
mM) was incubated with ROS (1 mM), NO
were treated with NAC (20 mM) or carboxyl-PTIO (50
mM) for 1 h,
solution (1 mM), GSH (200 mM), CuCl2 solution (10 mM), or FeCl3
solution (10 mM) at 37 ꢁC for 1 h, and the percentage of decom-
posed HQ-NO-11 was determined by RP-HPLC. To study the H2O2-
and then incubated with 12 M HQ-NO-11 for 24 h. Cells were
washed in ice-cold PBS, and fixed in ice-cold 70% alcohol for at least
24 h. After that, cells were washed twice in ice-cold PBS, resus-
m
mediated decomposition of HQ-NO-11, HQ-NO-11 (100
m
M) was
pended in 0.4 mL PBS and treated with 30 mL RNase A (1 mg/mL)
13

Y. Zhang, J. Yang, T. Meng et al.
European Journal of Medicinal Chemistry 212 (2021) 113153
(Thermo Scientific, MA, USA). Then 50
mL PI (500
mg/mL) (KGA105,
4.16. In vivo antitumor effects of HQ-NO-11
Keygentec, Nanjing, China) was added into cells to stain the cellular
DNA, and the staining process lasted 30 min at 4 ꢁC in darkness. The
DNA content of the stained cells was analyzed by flow cytometry
with a BD FACScalibur (BD Bioscience, Heidelberg, Germany), and
the cycle distribution was quantified.
Animal studies were approved by the Ethical Committee of
Jiangnan University and were performed according to the guide-
lines of the Laboratory Animal Management Regulations in China
and the Guide for the Care and Use of Laboratory Animals (National
Institutes of Health, revised 2011). Male BALB/c nude mice 3e5
weeks of age (Silaike Experiment Animal Co.,Ltd, Shanghai, China)
were inoculated subcutaneously with 1 ꢂ 107 SW1990 cells. After
the formation of a solid tumor with a volume of about 65 mm3, the
tumor-bearing mice were randomly divided into four groups with
eight mice in each group. Each group was intravenously injected
every three days with saline, 10 mg/kg HQ-NO-11, 20 mg/kg HQ-
NO-11, or 20 mg/kg 8-HQ, respectively. The tumor sizes were
monitored every two days for three weeks. At the end of the
experiment, the mice were sacrificed. The tumors were dissected,
4.13. Apoptosis assay
SW1990 cells were planted at 5 ꢂ 105 per well in 6-well plate
and incubated for 24 h. Cells were treated with indicated com-
pounds for 24 h, In case of NAC or carboxyl-PTIO pretreatment, cells
were treated with NAC (20 mM) or carboxyl-PTIO (50
and then incubated with 12 M HQ-NO-11 for 24 h. Cells were
collected and washed twice with ice-cold PBS, resuspended in
300
L 1 ꢂ binding buffer. Annexin V-FITC (KGA105, Keygentec,
China) (5 L) was added into each sample and incubated for
15 min at r.t. in darkness. PI (KGA105, Keygentec, China) (5 L) was
added into cells and stained for 5 min at r.t. in darkness. At last,
200
mM) for 1 h,
m
m
and the tumor sizes were calculated by the formula V
m
(mm3) ¼ d2 ꢂ D/2, where D is the largest diameter and d the
m
smallest diameter.
m
L 1 ꢂ binding buffer was added, and cellular apoptosis was
Declaration of competing interest
analyzed by using flow cytometry with a BD FACScalibur (BD
Bioscience, Heidelberg, Germany).
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.14. Cell mitochondrial membrane potential assay
Acknowledgements
SW1990 cells were planted at 5 ꢂ 105 per well in 6-well plate
and incubated for 24 h. Cells were treated with indicated com-
pounds for 24 h. In case of NAC or carboxyl-PTIO pretreatment, cells
This work is supported by the National Natural Science Foun-
dation of China (81602960, 81803349), Natural Science Foundation
of Jiangsu (BK20171048), the Fundamental Research Funds for the
Central Universities (JUSRP51712B, JUSRP12014), and National
First-class Discipline Program of Light Industry Technology and
Engineering (LITE2018-14).
were treated with NAC (20 mM) or carboxyl-PTIO (50
mM) for 1 h,
and then incubated with 12 M HQ-NO-11 for 24 h. The cells were
m
stained with the lipophilic cationic dye JC-1 (KGA602, Keygentec,
China) according to the manufacturer’s instruction. The percentage
of cells with healthy or collapsed mitochondrial membrane po-
tentials was monitored by flow cytometry with a BD FACScalibur
(BD Bioscience, Heidelberg, Germany).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113153.
4.15. Western blot assay
Abbreviations
SW1990 cells were planted at 5 ꢂ 105 per well in 6-well plate
and incubated at 37 ꢁC for 24 h. Then cells were treated with
indicated compounds for 24 h. In case of NAC or carboxyl-PTIO
pretreatment, cells were treated with NAC (20 mM) or carboxyl-
8-HQ
8-hydroxyquinoline;
amyloid
A
b
b
AD
PD
Alzheimer’s disease
Parkinson’s disease
PTIO (50
for 24 h.
mM) for 1 h, and then incubated with 12 mM HQ-NO-11
ROS
reactive oxygen species
hydrogen peroxide
hydroxyl radicals
peroxynitrite
nitric oxide
Cells were then placed on ice for 10 min, followed by two rounds
of centrifugation at 10000 rpm for 15 min, and protein concen-
tration was measured using a BCA (KGA902, Keygentec, China)
protocol. Protein was stored at ꢀ70 ꢁC until further use. SDS-PAGE
(KGP113, Keygentec, China) was used to separate the proteins and
then transferred to a nitrocellulose membrane. Membranes were
blocked for 1 h in 5% milk in Tris-buffered saline. Membranes were
incubated in antibodies, including anti-Cleaved Caspase3 antibody
(ab2302, Abcam, UK), anti-Cleaved Caspase9 antibody (ab2324,
Abcam, UK), anti-Bax antibody (ab32503, Abcam, UK), and anti-Bcl-
2 antibody (ab182858, Abcam, UK), overnight at 4 ꢁC. Following
three washes in TBST (1 ꢂ ) (NBP1-47398, KGP103X, Keygentec,
China), and then incubated with the secondary antibodies conju-
gated to horseradish peroxidase at r.t for 2 h. Membranes were
washed three times in TBST and then incubated in an enhanced
chemiluminescence (ECL) (KGP116, Keygentec, China) detection
substrate for 5 min. Densitometry of the appropriate bands was
performed using G: Box ChemiXR5 (Syngene, MD, USA).
H2O2
HO
ONOOꢀ
NO
O2ꢀ
superoxide
EDCI
HOBT
DIPEA
CLSM
3-NT
NAC
Djm
1-ethyl-3-(3-dimethylaminopropyl)
hydroxybenzotriazole
N,N-diisopropylethylamine
confocal laser scanning microscope
3-nitrotyrosine
N-acetylcysteine
membrane potential of mitochondria
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