Enhanced pyrazolopyrimidinones cytotoxicity against glioblastoma cells activated by ROS-Generating cold atmospheric plasma

Article abstract of DOI:10.1016/j.ejmech.2021.113736

Pyrazolopyrimidinones are fused nitrogen-containing heterocyclic systems, which act as a core scaffold in many pharmaceutically relevant compounds. Pyrazolopyrimidinones have been demonstrated to be efficient in treating several diseases, including cystic fibrosis, obesity, viral infection and cancer. In this study using glioblastoma U-251MG cell line, we tested the cytotoxic effects of 15 pyrazolopyrimidinones, synthesised via a two-step process, in combination with cold atmospheric plasma (CAP). CAP is an adjustable source of reactive oxygen and nitrogen species as well as other unique chemical and physical effects which has been successfully tested as an innovative cancer therapy in clinical trials. Significantly variable cytotoxicity was observed with IC₅₀ values ranging from around 11 μM to negligible toxicity among tested compounds. Interestingly, two pyrazolopyrimidinones were identified that act in a prodrug fashion and display around 5–15 times enhanced reactive-species dependent cytotoxicity when combined with cold atmospheric plasma. Activation was evident for direct CAP treatment on U-251MG cells loaded with the pyrazolopyrimidinone and indirect CAP treatment of the pyrazolopyrimidinone in media before adding to cells. Our results demonstrated the potential of CAP combined with pyrazolopyrimidinones as a programmable cytotoxic therapy and provide screened scaffolds that can be used for further development of pyrazolopyrimidinone prodrug derivatives.

Full text of DOI:10.1016/j.ejmech.2021.113736

European Journal of Medicinal Chemistry 224 (2021) 113736
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Enhanced pyrazolopyrimidinones cytotoxicity against glioblastoma
cells activated by ROS-Generating cold atmospheric plasma
, b, c, d,
Zhonglei He a
*, Clara Charleton e, Robert W. Devine e, Mark Kelada e,
John M.D. Walsh e, Gillian E. Conway a f, Sebnem Gunes a c, Julie Rose Mae Mondala a
,
,
c
,
,
, c
,
b,
,
, c
Furong Tian a c, Brijesh Tiwari g, Gemma K. Kinsella a c, Renee Malone a
,
,
,
Denis O'Shea a c, Michael Devereux a c, Wenxin Wang d, Patrick J. Cullen h,
,
, b, c, *
John C. Stephens e i, James F. Curtin a
a BioPlasma Research Group, School of Food Science and Environmental Health, Technological University Dublin, Dublin, Ireland
b Nanolab, FOCAS Research Institute, Technological University Dublin, Dublin, Ireland
c Environmental, Sustainability and Health Research Institute, Technological University Dublin, Dublin, Ireland
d Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin, Ireland
e Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
f In-Vitro Toxicology Group, Institute of Life Science, Swansea University Medical School, Swansea University, Singleton Park, Swansea, Wales, United
Kingdom
g Department of Food Biosciences, Teagasc Food Research Centre, Ashtown, Dublin, Ireland
h School of Chemical and Biomolecular Engineering, University of Sydney, Australia
i The Kathleen Lonsdale Institute of Human Health Research, Maynooth University, Maynooth, Co. Kildare, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolopyrimidinones are fused nitrogen-containing heterocyclic systems, which act as a core scaffold
in many pharmaceutically relevant compounds. Pyrazolopyrimidinones have been demonstrated to be
efficient in treating several diseases, including cystic fibrosis, obesity, viral infection and cancer. In this
study using glioblastoma U-251MG cell line, we tested the cytotoxic effects of 15 pyrazolopyrimidinones,
synthesised via a two-step process, in combination with cold atmospheric plasma (CAP). CAP is an
adjustable source of reactive oxygen and nitrogen species as well as other unique chemical and physical
effects which has been successfully tested as an innovative cancer therapy in clinical trials. Significantly
Received 11 May 2021
Received in revised form
28 July 2021
Accepted 29 July 2021
Available online 2 August 2021
Keywords:
variable cytotoxicity was observed with IC50 values ranging from around 11 mM to negligible toxicity
Cold atmospheric plasma
Pyrazolopyrimidinone
Pro-drug
ROS
Glioblastoma
among tested compounds. Interestingly, two pyrazolopyrimidinones were identified that act in a prodrug
fashion and display around 5e15 times enhanced reactive-species dependent cytotoxicity when com-
bined with cold atmospheric plasma. Activation was evident for direct CAP treatment on U-251MG cells
loaded with the pyrazolopyrimidinone and indirect CAP treatment of the pyrazolopyrimidinone in media
before adding to cells. Our results demonstrated the potential of CAP combined with pyrazolopyr-
imidinones as a programmable cytotoxic therapy and provide screened scaffolds that can be used for
further development of pyrazolopyrimidinone prodrug derivatives.
Programmable cytotoxicity
© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
heterogeneity, varied patient characteristics, treatment efficacy,
and toxicity considerations. A major limitation in treating advanced
Despite extensive efforts made in the battle against cancer over
the past decades, many challenges remain due to tumour
carcinoma is delivering sufficient concentrations of a chemother-
apeutic agent to the tumour site without causing undue toxicity
elsewhere. For this reason, prodrugs that can be locally activated at
the site of the tumour thereby having low toxicity elsewhere are of
particular interest.
* Corresponding authors. BioPlasma Research Group, School of Food Science and
Environmental Health, Technological University Dublin, Dublin, Ireland.
E-mail addresses: zhonglei.he@tudublin.ie (Z. He), james.curtin@tudublin.ie
(J.F. Curtin).
Reactive oxygen species (ROS), as a natural product generated
during normal metabolism pathways, play critical roles in various
https://doi.org/10.1016/j.ejmech.2021.113736
0223-5234/© 2021 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
cellular activities and are considered a carcinogenic factor as high
ROS levels are capable of inducing damage and mutation of inter-
cellular DNA and therefore causing malignancy [1]. However, as
understanding deepened, ROS was found to be a double-edged
sword for cancer cells [2]. Evidence showed that higher ROS
levels are generated in cancer cells than healthy cells, which is
attributed to the higher metabolic activities and more rapid pro-
liferation of transformed cells [2]. Hence, the cellular antioxidant
system works under a more significant load to protect tumour cells
from oxidative stress, a common feature in many types of tumours
that can be targeted to develop efficient therapies. Attempts have
been made to exploit this difference with healthy tissues, and
anticancer drugs have been developed that are conditionally acti-
vated by high levels of ROS [3]. Cold atmospheric plasma (CAP) can
locally induce ROS generation in cells and tissues with a high de-
gree of control of both the amount of ROS generated and the
location [4]. CAP treatment has been shown to induce remission of
tumour and promote wound healing for head and neck cancer
patients in a recent clinical trial [1]. Meanwhile, prodrugs, which
only have significant cytotoxicity after being activated by a high
ROS level, have fewer side effects and are more specific to cancer
cells [5]. Therefore, combining prodrugs with CAP to locally in-
crease tumour ROS and activate cytotoxicity only in tumour tissue
may provide a novel promising combination therapy against
cancer.
Pyrazolopyrimidinones have been identified with bioactivity for
the treatment of cancer [6], infections [7,8], obesity [9,10] and cystic
fibrosis [11,12]. It has also been reported that pyrazolopyr-
imidinones present notable inhibiting effects to the activity or
function of several kinases, including the PI3 kinase, glycogen
synthase kinase ꢀ3 (GSK-3), amongst others. These kinases are
involved and can play critical roles in various cellular activities,
including cell differentiation, motility, cell growth, proliferation,
survival and intracellular trafficking [13e16]. Within this initial
structure-activity relationship (SAR) study, 15 pyrazolopyr-
imidinones have been synthesised and screened for their potential
cytotoxic effects combined with CAP treatment, using the glio-
blastoma U-215MG cell line. Although the IC50 value of most
500 spectrometer or Bruker 300 Advance spectrometer at 293 K. All
chemical shifts were referenced relative to the relevant deuterated
solvent residual peaks or TMS. Assignments of the NMR spectra
were deduced using 1H NMR and 13C NMR, along with 2D experi-
ments (COSY, HSQC and HMBC). The following abbreviations were
used to explain the observed multiplicities; s (singlet), d (doublet), t
(triplet), q (quartet), hept (heptet), m (multiplet), bs (broad singlet),
pt (pseudo triplet). Flash chromatography was performed with
Merck Silica Gel 60. Microwave reactions were carried out using a
CEM Discover Microwave Synthesizer with a vertically focused
floor mounted infrared temperature sensor, external to the mi-
crowave tube. The 10 mL reaction vessels used were supplied from
CEM and were made of borosilicate glass. High resolution mass
spectrometry (HRMS) was performed on an Agilent-LC 1200 Series
coupled to a 6210 or 6530 Agilent Time-Of-Flight (TOF) mass
spectrometer equipped with an electrospray source in both positive
and negative (ESIþ/ꢀ) modes. Infrared spectra were obtained as
KBr disks in the region 4000e400 cmꢀ1 on a PerkinElmer Spectrum
100 FT-IR spectrophotometer. Further experimental details and
associated spectra can be found in the Supporting Information.
2.2. Synthesis of pyrazolopyrimidinones
Pyrazolopyrimidinones 1e4, 8, 9, 11, 12 were prepared as
described below, NMR spectra can be found in the Supporting In-
formation Figs. S1eS34. Pyrazolopyrimidinones 5e7, 10, 13, 14, 15
were prepared as per the literature procedure reported by Kelada
et al. with full experimental details and NMR spectra available in
the Supporting Information Fig. S1-S34 [11]. Full experimental
details and spectra for the synthesis of the required amino pyr-
azoles are also found in the Supporting Information.
2.2.1. Synthesis of 2-isopropyl-5-(4-nitrophenyl)pyrazolo[1,5-a]
pyrimidin-7(4H)-one (1)
Ethyl 3-(4-nitrophenyl)-3-oxopropanoate (209 mg, 0.88 mmol)
was heated at reflux with 3-amino-5-isopropylpyrazole (100 mg,
0.80 mmol) in acetic acid (6 mL) for 6 h. The reaction was cooled to
room temperature and the solvent removed under reduced pres-
sure. The residue was dissolved in tetrahydrofuran (THF) (~5 mL)
and petroleum ether (40e60 fraction, 5 mL) added. The resulting
precipitate was filtered and washed with a minimum of 1:1
mixture of THF: petroleum ether (40e60) to give 2-isopropyl-5-(4-
nitrophenyl)pyrazolo [1,5-a]pyrimidin-7(4H)-one, 83 mg (58%). 1H
cytotoxic candidate, compound 2, only reached around 11 mM and
the majority of them presented moderate, even marginal activity,
with or without CAP treatment, two leading prodrug candidates, 9
and 10, were identified. In combination with low dose CAP treat-
ment, which had negligible toxicity to cancer cells, the cytotoxicity
of the leading prodrug candidates 9 and 10 was activated and
enhanced by a factor of over 15 fold and 5 fold respectively (IC50
NMR: (300 MHz, CD3OD)
J ¼ 8.6 Hz, 2H), 6.19 (s,1H), 6.16 (s,1H), 3.12 (m,1H),1.37 (s, 3H),1.35
(s, 3H); 13C NMR: (75 MHz, CD3OD)
164.3, 157.5, 149.5, 148.8,
142.4, 138.7, 128.3, 123.8, 95.0, 86.6, 28.4, 21.4; HRMS: calcd for
15H15N4O3 m/z: [M
H]þ, 299.1139, found: 299.1149 [Diff
(ppm) ¼ 3.46].
d
8.44 (d, J ¼ 8.6 Hz, 2H), 8.05 (d,
against U-251MG cells decreased from around 940 to 62
mM, and
d
290 to 56 M, for 9 and 10 respectively). The combination cyto-
m
toxicity between 10 and CAP treatment has undergone further
investigation, where the ROS generated in culture medium by CAP
treatment has been determined to play the primary role in the
activation of prodrug 10. The CAP-induced modification of the
compounds are yet explored, but this work provides a potential
foundation for further exploration and development of more
effective prodrug derivatives based on the screened leading core
scaffolds.
C
þ
2.2.2. Synthesis of 5-(3,5-bis(trifluoromethyl)phenyl)-2-
butylpyrazolo[1,5-a]pyrimidin-7(4H)-one (2)
Ethyl
3-(3,5-bis(trifluoromethyl)phenyl)-3-oxopropanoate
(150 mg, 0.45 mmol) was heated at reflux with 3-butyl-1H-pyr-
azol-5-amine (63 mg, 0.45 mmol) in acetic acid (2 mL) for 6 h. The
reaction was cooled to room temperature and the solvent removed
under reduced pressure. The residue was dissolved in THF (~5 mL)
and petroleum ether (40e60 fraction, 5 mL) added. The resulting
precipitate was filtered and washed with a minimum of 1:1
mixture of THF: petroleum ether (40e60) to give 5-(3,5-
2. Materials and methods
2.1. Chemistry general information
All reagents for synthesis were bought commercially and used
without further purification. The 3-amino-5-isopropylpyrazole was
purchased from Fluorochem and used as received. Reactions were
monitored with thin layer chromatography (TLC) on Merck Silica
Gel F254 plates. NMR spectra were recorded using a Bruker Ascend
bis(trifluoromethyl)phenyl)-2-butylpyrazolo
[1,5-a]pyrimidin-
8.44 (s, 2H),
7(4H)-one, 43 mg (23%). 1H NMR (300 MHz, CD3OD)
d
8.24 (s, 1H), 8.32 (s, 1H), 6.22 (s, 1H), 6.18 (s, 1H), 2.78 (t, J ¼ 7.6 Hz,
2H), 1.74 (m, J ¼ 7.6 Hz, 2H), 1.44 (m, J ¼ 7.6 Hz, 2H), 0.98 (t,
J ¼ 7.6 Hz, 2H); 13C NMR (75 MHz, CD3OD)
d 158.7, 157.4, 147.9,
2

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
142.4, 135.3, 132.3 (q, JCF ¼ 34.1 Hz), 127.8 (m), 124.3 (m), 123.1 (q,
JCF ¼ 272 Hz), 95.1, 88.6, 31.2, 28.0, 22.0, 12.8.
reaction mixture was heated at reflux overnight. After cooling to rt,
the acetic acid was removed under reduced pressure and tritura-
tion with ethyl acetate (5 mL) gave 2,5-di-tert-butylpyrazolo [1,5-a]
pyrimidin-7(4H)-one, 42 mg (23 %). 1H NMR: (300 MHz, CD3OD)
2.2.3. Synthesis of 2-isopropyl-5-(4-methoxyphenyl)pyrazolo[1,5-
a]pyrimidin-7(4H)-one (3)
d
6.08 (s, 1H, H5), 5.74 (s, 1H, H2), 1.38 (s, 18H, (CH3)3 ꢂ 2); 13C NMR:
A microwave tube was charged with ethyl 4-methoxybenzoyl
acetate (0.17 mL, 0.9 mmol), 3-amino-5-isopropylpyrazole
(110 mg, 0.9 mmol), and methanol (2 mL) and subjected to mi-
crowave irradiation (100 W, 120 ꢁC) for 1 h. At this point, three
drops of acetic acid were added, and the reaction mixture subjected
to microwave irradiation (100 W, 120 ꢁC) for a further 2 h. The
resultant precipitate was removed by filtration and washed with a
minimum amount of cold methanol, giving 42 mg of 2-isopropyl-5-
(125 MHz, CD3OD) d 166.7, 161.6, 158.5, 142.2, 91.3, 85.4, 34.9, 32.4,
29.2, 27.6; Rf: 0.6 (1:9 v/v, MeOH:DCM); HRMS: calcd for C14H22N3O
m/z, [M þ H]þ, 248.1757, found: 248.1768, [Diff (ppm) ¼ 4.45]; IR
(KBr): 3090 (NeH), 2965 (CeH), 1670 (C]O), 1614 (C]C), 1570
(NeH), 1481 (C]C), 1240 (CeN) cmꢀ1
.
2.2.7. Synthesis of 5-(3,5-dimethylphenyl)-2-methylpyrazolo[1,5-a]
pyrimidin-7(4H)-one (11)
(4-methoxyphenyl)pyrazolo
[1,5-a]pyrimidin-7(4H)-one.
The
Ethyl 3-(3,5-dimethylphenyl)-3-oxopropanoate (0.56 mL,
3.0 mmol) was added to a solution of 3-amino-5-methylpyrazole
(132 mg, 1.0 mmol) dissolved in acetic acid (5 mL) and heated at
reflux for 21 h. Volatiles were removed under reduced pressure.
The product was purified by column chromatography (100% DCM)
to give 5-(3,5-dimethylphenyl)-2-methylpyrazolo [1,5-a]pyr-
imidin-7(4H)-one, 204 mg, (59 %). 1H NMR: (500 MHz, CD3OD)
filtrate was evaporated to dryness and the resulting residue
recrystallised from methanol to give a further 85 mg of 2-isopropyl-
5-(4-methoxyphenyl)pyrazolo [1,5-a]pyrimidin-7(4H)-one. The
combined yield was 127 mg (50%). 1H NMR: (300 MHz, DMSO‑d6)
d
12.21 (s, 1H, NH), 7.79 (d, J ¼ 8.9 Hz, 2H), 7.13 (d, J ¼ 8.9 Hz, 2H),
6.03 (s, 1H), 5.95 (s, 1H), 3.85 (s, 3H), 3.01 (m, 1H), 1.28 (s, 3H), 1.26
(s, 3H); 13C NMR: (75 MHz, DMSO‑d6)
161.8, 161.4, 156.3, 142.1,
d
d
7.42 (s, 2H), 7.26 (s, 1H), 6.13 (s, 1H), 6.05 (s, 1H), 2.53e2.34 (m,
128.7, 114.4, 92.5, 86.4, 55.5, 27.9, 22.3; HRMS: calcd for C16H18N3O
9H); 13C NMR: (126 MHz, CD3OD)
d
138.9, 132.3, 124.5, 19.9; HRMS:
m/z: [M þ H]þ, 284.1394, found: 284.1388 [Diff (ppm) ¼ 2.05].
calcd for C13H16N3O m/z, [M þ H]þ, 254.1293’ found: 253.1207, [Diff
(ppm) ¼ 3.3]; Rf: 0.11 (100 % DCM); IR (KBr): 3251 (NeH), 2917
2.2.4. Synthesis of 5-(3,5-bis(trifluoromethyl)phenyl)-2-
isopropylpyrazolo[1,5-a]pyrimidin-7(4H)-one (4)
(CeH), 1665 (C]O), 1618 (NeH), 1597 (Ar CeC) cmꢀ1
.
A microwave tube was charged with 3-isopropyl-1H-pyrazol-5-
amine (113 mg, 0.9 mmol), ethyl 3-(3,5-bis(trifluoromethyl)
2.2.8. Synthesis of 5-(3,5-dimethylphenyl)-2-isopropylpyrazolo[1,5-
a]pyrimidin-7(4H)-one (12)
phenyl)-3-oxopropanoate (295 mg, 0.9 mmol), acetic acid (28.6
mL,
Ethyl 3-(3,5-dimethylphenyl)-3-oxopropanoate
(28
mL,
0.5 mmol), MeOH (1 mL), and subjected to MW irradiation (100 W,
150 ꢁC) for 2 h. The resulting mixture was concentrated in vacuo
and purified by trituration with cold ethyl acetate to give 5-(3,5-
bis(trifluoromethyl)phenyl)-2-isopropylpyrazolo [1,5-a]pyrimidin-
1.4 mmol) was added to a solution of 3-isopropyl-1H-pyrazol-5-
amine (176 mg, 1.4 mmol) dissolved in acetic acid (5 mL) and
refluxed for 5.5 h. Volatiles were removed under reduced pressure.
The product was purified by trituration using ethyl acetate (10 mL)
and give 5-(3,5-dimethylphenyl)-2-isopropylpyrazolo [1,5-a]pyr-
imidin-7(4H)-one, 165.4 mg (42 %). 1H NMR: (500 MHz, CDCl3)
7(4H)-one, 140 mg (40%). 1H NMR (500 MHz, DMSO‑d6)
d 12.65 (bs,
1H), 8.51 (s, 2H), 8.32 (s, 1H), 6.30 (s, 1H), 6.10 (s, 1H), 3.05e2.95 (m,
J ¼ 13.5, 6.7 Hz, 1H), 1.25 (d, J ¼ 6.8 Hz, 6H); 13C NMR (126 MHz,
d
9.51 (bs, 1H), 7.25 (s, 2H), 7.13 (s, 1H), 6.00 (m, 2H), 3.10 (septet,
DMSO‑d6)
d
162.4, 156.1, 146.3, 142.1, 135.0, 131.1 (q, JCF ¼ 33.3 Hz),
1H), 2.34 (s, 6H), 1.28 (d, J ¼ 7.0 Hz, 6H); 13C NMR: (126 MHz, CDCl3)
128.5, 124.4, 123.2 (q, JCF ¼ 273.4 Hz), 95.5, 86.9, 28.0, 22.3; Rf 0.5
(1:9 v/v MeOH:EtOAc); HRMS: calcd for C17H14F6N3O m/z, [M þ
H]þ, 390.1036, found: 390.1051, [Diff (ppm) ¼ 2.79]; IR (KBr): 3423
(NeH), 2972 (CeH), 1670 (C]O), 1617 (C]C), 1577 (NeH), 1481
d 157.8, 141.6, 139.1, 132.8, 132.7, 124.5, 94.6, 86.7, 28.6, 22.5, 21.3;
HRMS: calcd for C17H20N3O m/z: [M þ H]þ, 281.1528, found:
281.1528 [Diff (ppm) ¼ 0.06]; IR (KBr): 3199 (NeH), 3081 (CeH),
1651 (C]O), 1595 (NeH), 1291 (Ar CeN) cmꢀ1
.
(C]C), 1279 (CeF), 1139 (CeN) cmꢀ1
.
2.3. Cell culture
2.2.5. Synthesis of 2-isopropyl-6-methyl-5-phenylpyrazolo[1,5-a]
pyrimidin-7(4H)-one (8)
U-251 MG (formerly known as U373MG) (ECACC 09063001),
human brain glioblastoma cancer cells (Obtained from Dr Michael
Carty, Trinity College Dublin) and HEK293, human embryonic kid-
ney cells (Obtained from Dr Darren Fayne, Trinity College Dublin)
were cultured in DMEM-high glucose medium (Merck, D6429)
supplemented with 10 % FBS and maintained in a 37 ꢁC incubator
within a humidified 5 % (v/v) CO2 atmosphere. For prodrug treat-
ment, DMEM-high glucose medium without pyruvate (Merck,
D5796) was used to make up culture medium to avoid the anti-
oxidant effects of pyruvate [17].
Ethyl
2-methyl-3-oxo-3-phenylpropanoate
(125
mg,
0.61 mmol) was heated at reflux with 3-amino-5-
isosubstitutedpyrazole (69 mg, 0.55 mmol) in acetic acid (2 mL)
for 15 h. The reaction was allowed to cool to room temperature and
the solvent removed under reduced pressure. The residue was
triturated with ethyl acetate to yield a solid. The solid was isolated
by filtration, washed with ethyl acetate, and dried in a drying pistol
to give 2-isopropyl-6-methyl-5-phenylpyrazolo [1,5-a]pyrimidin-
7(4H)-one, 54 mg (37%). 1H NMR (500 MHz, MeOD)
d 7.70e7.45 (m,
5H), 5.98 (s, 1H), 3.08 (hept, J ¼ 7.0 Hz, 1H), 2.00 (s, 3H), 1.32 (d,
J ¼ 7.0 Hz, 6H). 13C NMR (126 MHz, MeOD)
d
164.0, 163.9, 158.7,
2.4. H2DCFDA assay
148.0, 141.6, 141.5, 133.8, 129.8, 128.5, 128.4, 101.7, 101.7, 84.7, 28.4,
28.4, 21.6, 10.7; HRMS: calcd for C16H18N3O m/z, [M þ H]þ,
268.1444, found: 268.1452, [Diff (ppm) ¼ 2.95].
H2DCFDA was used to detect ROS induced by CAP treatment. U-
251 MG cells were seeded into 35 ꢂ 10 mm tissue culture dishes
(Sarstedt) at a density of 2 ꢂ 105 cells/ml, or 35 mm glass-bottom
dishes (Greiner Bio-One) at a density of 1 ꢂ 105 cells/ml and
incubated overnight as above to allow adherence. Prior to treat-
ment with CAP at 75 kV for 30 s, cells were first incubated with
2.2.6. Synthesis of 2,5-di-tert-butylpyrazolo[1,5-a]pyrimidin-
7(4H)-one (9)
The amino pyrazole 5-amino-3-(tert-butyl)-1H-pyrazole
(100 mg, 0.71 mmol) was dissolved in acetic acid (10 mL), and ethyl
25
mM H2DCFDA (Thermo Fisher Scientific) in serum-free medium
4,4-dimethyl-3-oxopentanoate (116
mL, 0.65 mmol) added. The
for 30 min at 37 ꢁC, then washed with phosphate buffered saline
3

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
(PBS) twice, culture medium once. Afterwards, cells were observed
under a Zeiss LSM 510 confocal laser scanning microscope (exci-
tation 488 nm, emission 505e530 nm), or were collected for
measurement with flow cytometry.
for indicated hours before Alamar Blue assays.
2.6. Cytotoxicity and inhibitor assays
Cell viability was analysed using the Alamar blue assay (Thermo
Fisher Scientific). 48 h after CAP treatment, the cells were rinsed
once with PBS, incubated for 3 h at 37 ꢁC with a 10 % Alamar blue/
90 % culture medium solution. The fluorescence was then measured
(excitation, 530 nm; emission, 595 nm) by a Victor 3 V 1420
microplate reader (PerkinElmer). 4 M N-Acetyl Cysteine (NAC)
stock solution was prepared in water. 4 M NAC solution was then
diluted in culture medium to a final concentration of 4 mM before
the addition of prodrugs and CAP treatment. The CAP treated cul-
ture medium containing prodrugs and 4 mM NAC was then added
into 96-well plates to incubate with U-251 MG cells.
2.5. CAP configuration and prodrug treatment
An experimental atmospheric dielectric barrier discharge (DBD)
plasma reactor, DIT-120, was used which has been described and
characterised in detail elsewhere [18,19]. Unless otherwise stated,
all U-251 MG cells were treated within 96-well plates. During CAP
treatment, containers were placed in between two electrodes, at a
voltage level of 75 kV for 10e40 s. The culture medium was
removed before CAP treatment then replaced with fresh culture
medium immediately afterwards.
An optimised prodrug treatment protocol was developed and
applied, as described below. U-251 MG cells were plated into 96-
2.7. Live/dead staining
well plates (Sarstedt) at a density of 1 ꢂ 104 cells/well (100
ml
standard culture medium per well) and were incubated overnight
for proper adherence. Unless otherwise stated, the plating map was
6 ꢂ 10 wells, for ten different concentrations, negative and positive
control groups (5 replicates for each group). All prodrugs were
dissolved and prepared in 100 % DMSO for the stock solution, which
was then serially diluted in culture medium to different concen-
U-251MG cells were seeded in 96-well plate at a density of
1 ꢂ 104 cells/well and allow overnight for adherent, as described
above. Cells were then treated with 2000, 290 and 0.3 mM of 10
respectively in culture medium without pyruvate for 48 h. The live/
dead staining was performed with LIVE/DEAD™ Viability/Cyto-
toxicity Kit, for mammalian cells (ThermoFisher). In brief, after 48-
h incubation with compounds, the previous medium was removed
trations as indicated. 10 ml of prodrug non-pyruvate culture me-
dium solution was then added to each well after removing the
previous medium, and the remainder was added into empty wells
in the plate. The plate then was treated with CAP at 75 kV for
and a staining PBS solution containing 2
mM of Calcein-AM and
4
mM of ethidium homodimer-1 (EthD-1) was added and incubated
at room temperate for 30 min, prior to fluorescent imaging (exci-
10e40 s as indicated. 90
solution was then added to corresponding wells with cells to final
volume 100 l immediately after CAP treatment. Cell viability as-
m
l of CAP-treated prodrug culture medium
tation/emission: Calcein-AM 494/517 nm; EthD-1528/617 nm).
m
2.8. Statistical analysis
says were then carried out following incubation for the indicated
time. Temozolomide (TMZ) (Merck) was dissolved in DMSO as stock
(50 mM) and then subsequently diluted in culture medium to
concentrations as indicated. U-251 MG cells were plated into 96-
well plates at a density of 2 ꢂ 103 cells/well and were incubated
overnight to ensure proper adherence. The combination treatment
of TMZ and 30 s CAP was performed with the same method as
above. Meanwhile, the TMZ stock was first treated with CAP for
15 min then diluted in culture medium to indicated concentrations
and incubated with untreated cells, for six days, as indicated.
Indirect CAP treatment was used to investigate the effects of CAP
on pro-drugs alone to determine the mechanism of combination
cytotoxicity to U-251 MG cells. Pro-drugs were treated with CAP at
75 kV for 10 s to 10 min in DMSO stock solution or culture medium
solution as indicated. The CAP-treated prodrug solution was then
added to U-251 MG cells in 96-well plates to incubate with U-251
MG cells. For CAP-activated medium, the culture medium was
treated with CAP at 75 kV for 30 s in 96-well plate and incubated
overnight at 4 ꢁC to remove short-lived reactive species. The pro-
drug stock solution was then serially diluted in the overnight
storage CAP-activated medium and incubated with U-251 MG cells.
Triplicate independent tests were carried out for each data point
unless indicated otherwise. Error bars of all figures are presented
using the standard error of the mean (S.E.M). Prism 7 (GraphPad
Software, San Diego, CA, USA) was used to carry out curve fitting
and statistical analysis. The isobologram was carried out using
CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA, www.
combosyn.com) [20]. Two-tailed
P values were used where
alpha ¼ 0.05. The significance between data points was verified
using one-way ANOVA and two-way ANOVA with Tukey's multiple
comparison post-test, as indicated in figures (*P < 0.05, **P < 0.01,
***P < 0.001, ****p < 0.0001).
3. Results
3.1. ROS generation by low dose CAP treatment
CAP has been well known for inducing the generation of ROS
[19,21,22]. In addition to flow cytometry, we have used a confocal
microscope and ROS indicator H2DCFDA to demonstrate the ROS
generated by 30 s of CAP treatment (Fig. 1A and B). 30 s of CAP
treatment is a small dose presenting relatively low cytotoxicity to
U-251 MG cell lines, with only a decrease of around 20 % in cell
viability as previously described [19,21]. As seen in Fig. 1C, 0-40 s of
CAP treatment decreased around 10e20 % of cell viability when U-
251 cells were treated and incubated in the culture medium
without pyruvate. TMZ, the standard chemotherapy for glioblas-
toma, has been tested in combination with 30 s and 15 min of CAP
treatment on cells or TMZ stock solution separately. CAP has pre-
viously been demonstrated to restore drug sensitivity in cisplatin-
resistant ovarian cancer cells [23], paclitaxel-resistant breast can-
cer cells [24], tamoxifen-resistant breast cancer cells [25], and
temozolomide-resistant malignant glioma cells [26]. As seen in
Fig. 1D, there is a marginal decrease of IC50 values of TMZ when U-
For CAP-activated cells, the U-251 MG cells overlaid with 10
ml
culture medium were treated with CAP for 30 s in 96-well plates,
while remainder of culture medium was added into empty wells in
the plate. Then the CAP-treated culture medium was added to each
well to 100 ml. Those CAP-activated cells were then incubated in the
CAP-activated medium for 0e5 h as indicated. Following incuba-
tion, the medium was replaced with fresh medium containing
corresponding concentrations of prodrugs. ROS generator, 2,20-
azobis (2-amidinopropane) dihydrochloride (AAPH, purchased
from Merck), was dissolved in water to 0.5 M as stock solution and
kept at ꢀ20 ꢁC till use. The AAPH stock solution was diluted in
pyruvate-free culture medium to desired concentrations then
incubated with HEK293 or U-251MG cells combined with prodrugs
4

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
Fig. 1. Treatment of U-251 MG cells with reactive species-generating CAP treatment alone or in combination with TMZ. (A) Fluorescence levels of oxidised H2DCFDA in
untreated and 30s CAP treated cells were observed via confocal microscope. (B) Fluorescence level of intracellular oxidised H2DCFDA was measured in U-251 MG cells via Flow
cytometry, left curve (green, untreated cells), right curve (red, 30 s CAP treated cells). (C) U-251 MG cells were treated with CAP for 10e40 s at 75 kV alone and then incubated for
48 h before cell viability was assessed. (D) CAP untreated or CAP 30s treated U-251 MG cells were incubated with a serial dilution of TMZ only or 15min CAP treated TMZ stock
solution for 6 days, Cell viability assays were then carried out, and IC50 values were calculated using GraphPad Prism.
251 MG cells were treated with both TMZ and 30s of CAP treatment,
which is in agreement with previously published studies demon-
strating that CAP possesses an ability to restore the sensitivity of U-
251 MG cells to TMZ [19,26]. We also found that 15 min of CAP
treatment on TMZ stock solution did not induce a significant
degradation of TMZ cytotoxicity. Instead, we observed a similar
marginal decrease in IC50 values (Fig. 1D and Supporting Informa-
tion Fig. S35).
On the other hand, some pyrazolopyrimidines were found to be
involved in cellular ROS generation. (4-Amino-6-hydroxypyrazolo-
3,4-d-pyrimidine) was found to inhibit xanthine oxidase and
decrease the enzymatic formation of uric acid and ROS [27].
Another pyrazolopyrimidine was demonstrated to generate ROS in
NCIeH460 (non-small-cell lung cancer) cells, leading to a loss in
mitochondrial membrane potential and apoptosis of the cancer
cells [28]. Therefore, in this initial SAR study, 15 pyrazolopyr-
imidinones were selected to study their cytotoxicity against U-251
MG cells, and more specifically the possible prodrug activation ef-
fects with ROS generating CAP treatment.
CAP treatment in order to identify possible candidates that are
activated by CAP (via restoring cellular sensitivity or a ROS-
dependent prodrug effect). As seen in Fig. 2, most pyrazolopyr-
imidinones had no activated cytotoxicity when the compounds,
together with cells in media, were directly treated by CAP for 30 s in
media. The dose-response curves of CAP-treated groups presented
high comparability and similar trends with untreated groups, as
30 s of CAP treatment presents negligible or low cytotoxicity when
applied alone. On the other hand, two compounds from the pyr-
azolopyrimidinone family, 9 and 10, presented significant activated
cytotoxicity in combination with 30 s of direct CAP treatment
(Fig. 2). The CAP treatment induced a pronounced descent of dose-
response curves with increasing compound concentration. As seen
in Table 1, CAP treatment induced significant decreases of IC50
values of 9 (from 940.6
mM to 62.41 mM) and 10 (from 292.8 mM to
56.79 M) while presenting low cytotoxicity by themselves.
m
Therefore, among all 15 candidates, 9 and 10 were determined as
the leading candidates with potential as CAP-activated drugs.
One can notice that the 95 % confidence intervals for of com-
pounds 5 (both groups) and 9 (control groups) were large. This is
due to that the cell viability of upper end of the dose ranges was still
lower than 50 %, demonstrating their limited activity against glio-
blastoma cell lines. Thus, the IC50 value was calculated via non-
linear regression using GraphPad Prism, which gave large 95 %
confidence intervals in this case. The dose ranges were limited by
the solubility of compounds in DMSO, as well as the limited dilution
of DMSO stock in culture media.
3.2. Screening of pyrimidone bicycle family compounds
As described in the Methods section and Supporting Informa-
tion 15 pyrazolopyrimidinone compounds were synthesised in a
two-step process, via an aminopyrazole intermediate, from the
corresponding keto nitrile and keto ester (Fig. 5). The cytotoxicity of
the compounds was evaluated in combination with small doses of
5

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
6

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
3.3. Plasma activated media enhances cytotoxicity of leading pro-
drug candidate
Our results demonstrate that 10 was activated by reactive spe-
cies generated during CAP treatment, therefore presenting signifi-
cant activated cytotoxicity to U-251 MG cells when combined with
CAP treatment. Furthermore, overnight storage CAP-activated me-
dium and CAP-activated cells have been used to study the dose-
response of U-251 MG cells to 10. When cells were treated with
overnight storage CAP-activated medium and prodrug, no activa-
tion was observed (Supporting Information Fig. S37b). Meanwhile,
when the U-251 MG cells were incubated for 0 he5 h after CAP
treatment, then incubated with fresh medium containing prodrug,
there was no significant activation of the prodrug observed, either
(Supporting Information Fig. S37c). In addition, a ROS generator,
AAPH, was used to test its potential activation effect towards 10.
AAPH is a stable compound that constantly generates peroxyl
radicals (ROOꢃ) at 37 ꢁC in aqueous solution for several hours and is
considered to induce reliable oxidative stress model [31,32].
Interestingly, in U-251MG and HEK293 cell lines, the addition of
3 mM AAPH in pyruvate free culture medium, which only decrease
20 % cell viability alone, significantly decreased cell viability to 50 %
combined with low concentrations of 10. However, the cytotoxicity
We selected 10 to investigate further the prodrug activated
cytotoxic effects combined with CAP treatment. Live/dead staining
was performed to further determine the status of cells in addition
to Alamar Blue assay. After incubation with 0.3, 290 and 2000 mM of
10 for 48 h, around 100 %, 50 % and 10 % cell viability was deter-
mined by Alamar Blue assay. As seen in Fig. 3, a significant decrease
of living cells stained with green (Calcein-AM) and a moderate
increase of dead cells stained with red (ethidium homodimer-1,
EthD-1) was observed from 100 % to 10 % groups. Also, distinct
morphological changes was demonstrated in 10 % cell viability
group. Thus,10 may decrease the cell viability via cytotoxicity along
with inhibition of proliferation, in which the cell death pathways
are awaiting further exploration. In addition, the cytotoxicity of 10
against HEK293 cells was tested compared to U-251MG cells, and a
slightly higher cytotoxic effect in HEK293 cells (IC50,123.3
mM, 95 %
confidence intervals, 111.5e136.5
mM) was observed (Supporting
Information Fig. S36).
On the other hand, the prodrug activation mechanism was
investigated. 10 was first dissolved in 100 % DMSO to 200 mM and
then treated with 30 s and 10 min of CAP. Afterwards, CAP-
treatment 10 DMSO solution was diluted in the culture medium
to incubate with cells. As seen from Fig. 4A and Table 2, no signif-
icant increase in cytotoxicity was observed whether the 10 DMSO
solution was treated with CAP or not. ROS generation is absent in
100 % DMSO solvent, whereas the generation of reactive species
relies on the presence of reactants in treated liquid [29], and DMSO
may function as a scavenger of OH radicals [30]. Therefore, we
hypothesised that ROS generation might be essential for the acti-
vation of compound 10.
10 was then diluted in culture medium first and treated with
CAP in 10 s increments from 0 s to 40 s. The CAP-treated 10 culture
medium solution was subsequently incubated with U-251 MG cells
for 48 h before the Alamar Blue assay. As seen in Figs. 1C and 4B and
Table 3, CAP 10e40 s treated 10 culture medium solution induced a
pronounced decrease of cell viability with concentrations higher
of 10 was not enhanced when the concentration is above 200 mM
(Supporting Information Figs. S36a, b, e). When the prodrug 10 was
co-incubated with 0.5 mM of AAPH, no enhanced cytotoxicity was
observed in both cell lines (Supporting Information Figs. S36c and
d).
4. Discussion and conclusions
Despite improvements in precision surgery, targeted and
focused chemotherapy and radiotherapy, emerging immuno-
therapy, and quick and accurate diagnosis of tumours, cancer
mortality remains a significant global health burden, accounting for
almost 10 million deaths in 2018, or 1 in 6 of all mortalities [33].
Current primary treatments still present several side effects when
applied alone with high doses, including poor patient experience,
secondary malignancy and
a variety of long-term sequelae
[2,34e36]. In this case, combination therapy has been considered a
promising approach to target tumours while generating fewer side
effects.
than 10 mM compared to cells treated with media not exposed to
CAP. CAP treatment of media alone had low cytotoxicity (~20 %
decrease in cell viability, Fig.1C). Meanwhile, NAC was applied as an
antioxidant to investigate the mechanism further. The 4 M NAC
solution was diluted in the culture medium to 4 mM before adding
10 and 40 s CAP treatment. As seen in Fig. 4B and Table 3, the NAC
and CAP treated group presented much higher cell viability and a
higher IC50 value than the CAP treated groups, which demonstrated
that ROS generated in culture media by CAP treatment played an
essential role in activating the effect of 10 against U-251MG cells.
Using CompuSyn software, the normalised isobologram was ana-
lysed, and the combination index (CI) values have been calculated
to evaluate the combination treatment. The CI values of 10 were
0.31099 with 10 s, 0.33520 with 20 s, 0.47561 with 30 s, and
0.57796 with 40 s of CAP treatment, which were all less than 1.00
and confirmed the significant prodrug activation effect (Fig. 4C). 11,
which is structurally similar to compound 10, two meta methyl
substituents added to the pheny ring, was also tested for compar-
ison. Interestingly, as seen in Supporting Information Fig. S37a, 11
showed no enhanced cytotoxicity when treated in culture medium
with CAP for 10e40 s, which suggests an important role for the
phenyl ring of the pyrazolopyrimidinone.
With the altered metabolism and rapid proliferation, the high
ROS level in cancer cells is considered a promising target for
developing specific therapies [37]. The chemotherapy agents
related to ROS can be divided into ROS-trigger drugs and ROS-
activated drugs. ROS-trigger drugs can further increase the ROS
level in cancer cells beyond their tolerable allowance to trigger cell
death, such as fenretinide [38], nitric oxide-donating aspirin [39],
imexone [40] and motexafin gadolinium [41]. On the other hand,
ROS-activated drugs, also termed prodrugs, can be activated and
become cytotoxic by the relative high ROS level in cancer cells,
leading to specific cancer cell killing effects. Meanwhile, CAP, a
novel technology allows a localised generation of ROS at a tumour
site, has been demonstrated to have significant therapeutic po-
tential in cancer treatment [1,42e44]. In this case, CAP provides the
ideal mechanism to augment or activate ROS-sensitive pro-drugs in
targeted areas.
Modifying bioactive scaffold cores with functional groups or
linkers that can be specifically activated and altered is a common
strategy to develop prodrugs. A novel imidazopyridazine, with a
sulfoxide/sulfone prodrug-like strategy, was found to cure mice
infected with P. berghei parasites at 4 ꢂ 50 mg/kg, when the
Fig. 2. Dose response curves of Pyrazolopyrimidinones. The stock solution of pyrazolopyrimidinones 1e15 were prepared in 100 % DMSO, which was then serially diluted in culture
medium without pyruvate to different concentrations as indicated. U-251 MG cells were treated with 1e15 in combination with 30 s of CAP treatment as indicated in the Methods
section. Cell viability assays were then carried out following incubation with 1e15 culture medium solution for 48 h.
7

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
Table 1
IC50 values and 95 % confidence intervals of pyrazolopyrimidinones alone or in combination with 30 s of CAP treatment against U-251 MG cells.
Control CAP 30 s
IC50 M) IC50 M)
Compound number
(
m
Hillslopes
ꢀ0.9429
ꢀ1.121
95 % Confidence Intervals (
22.84 to 28.50
10.09 to 12.70
174.3 to 222.7
37.75 to 48.74
5733 to 31,190
66.55 to 90.67
306.1 to 439.5
120.2 to 156.6
564.2 to 1568
mM)
(
m
Hillslopes
ꢀ1.125
ꢀ1.159
ꢀ1.918
ꢀ0.5718
ꢀ1.007
ꢀ0.5991
ꢀ0.3780
ꢀ1.100
ꢀ1.741
ꢀ2.198
95 % Confidence Intervals (
11.53 to 14.48
10.34 to 12.57
113.0 to 142.4
13.75 to 26.38
1831 to 17,555
60.59 to 89.44
197.3 to 1082
mM)
1
2
3
4
5
6
7
8
25.51
11.32
197
42.9
13,372
77.68
366.8
137.2
940.6
292.8
Not converged
502.2
23.34
74.59
148.1
12.92
11.4
ꢀ1.149
126.8
19.04
5670
73.62
462.1
86.27
62.41
56.79
Not converged
608.6
38.23
44.42
86.7
ꢀ1.043
ꢀ0.5624
ꢀ0.6622
ꢀ1.649
ꢀ0.8449
ꢀ0.9807
ꢀ1.344
76.20 to 97.68
49.58 to 78.56
50.93 to 63.33
9
10
11
12
13
14
15
252.2 to 340.0
ꢀ1.201
ꢀ1.101
ꢀ0.8717
ꢀ1.340
452.4 to 557.4
19.82 to 27.49
67.68 to 82.20
131.7 to 166.6
ꢀ1.432
ꢀ1.475
ꢀ0.5734
ꢀ1.406
561.0 to 660.2
34.96 to 41.80
31.39 to 62.85
75.02 to 100.2
Fig. 3. Live/dead staining of U-251MG cells. ~100 %, 50 % and 10 % cell viabilities were achieved by inucabating cells with 0.3, 290 and 2000 mM of compound 10 for 48 h.
methylsulfoxide derivative rapidly generated the methylsulfone
derivative in vivo, leading to higher plasma exposure compared to
direct use of the methylsulfone derivative [45]. Hybrids of two
active compounds via a prodrug linker has also been exploited as
combination treatments. Rapamycin derivatives have been conju-
gated with hydroxywortmannin via linkers like diester bond for
potential synergistic efficacy of concomitant inhibition of PI3K and
mTOR for cancer treatment [46].
On the other hand, many pyrazolopyrimidines have found use in
drug development and have potential in antiviral, antimicrobial,
anticoccidials, antitumour and anti-inflammatory activities [47].
Pyrazolopyrimidine derivatives have been found to inhibit many
protein kinases, such as Src kinase [48], cyclin-dependent kinases
[49,50], Chk1 Checkpoint kinase [51], B-Raf protein kinase [52] and
Aurora-A kinase [53], which are considered significant targets for
anticancer drug development, as the alterations in many kinase
activities are involved in cancerous mutations [54]. The potential
interactions between this diversified family and ROS have also been
noted [27,28].
activation effects combined with ROS-generating CAP treatment.
The pyrazolopyrimidinones were synthesised using a two-step
process (Fig. 5), which involved generating an isolatable amino
pyrazole intermediate (step one) and its subsequent reaction with
the corresponding
b-ketoester to give the desired pyrazolopyr-
imidinone (step two) [11]. A family of pyrazolopyrimidinones was
generated, which incorporated structural variation at R1
(R1 ¼ phenyl, substituted aryl, or alkyl groups) and R2 (R2 ¼ phenyl,
substituted aryl, or alkyl groups). The pyrazolopyrimidinones were
generated in 16e67 % yields and underwent structural charac-
terised using NMR spectroscopy, IR spectroscopy, and mass spec-
trometry (see Method section and Supporting Information). The
majority of candidates showed moderate to marginal cytotoxic ef-
fect towards tumour cells when applied alone or in combination
with 30 s of CAP treatment. Although the IC50 values presented no
significant cytotoxicity alone or promising specific tumour cell-
killing efficacy (Table 1, Fig.
2 and Supporting Information
Fig. S36), we identified two compounds, 9 and 10, presenting sig-
nificant prodrug activation effects. Low doses of CAP treatment
with negligible cytotoxicity were demonstrated to significantly
enhance the cytotoxic effect of 9 and 10 by more than 15 and 5
Therefore, 15 pyrazolopyrimidinones, a class of pyrazolopyr-
imidine derivatives, were selected to study their possible prodrug
8

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
Fig. 4. Dose responses of U-251 MG cells to 10 treated by CAP in culture medium solution or DMSO stock solution. (A) 10 was prepared in DMSO stock solution and treated with
CAP for 30 s and 10 min, serially diluted into fresh culture medium and incubated with U-251 MG cells for 48 h and compared with control groups. (B) 10 was prepared in the
culture medium, treated with CAP for 0e40 s with or without NAC and then incubated with U-251 MG cells in 96-well plates for 48 h before cell viability was assessed. (C)
Isobologram analysis of the combinational effect of 10 and CAP. The single doses CAP on the y-axis and 10 on the x-axis were used to draw the additivity line. Four combination
points were indicated in the isobologram (CAP 10e40s and corresponding IC50 values of CAP-treated 10). The localisation of combined 10 and CAP at different time exposures can be
translated to synergism CI < 1, additivity CI ¼ 1 or antagonism CI > 1.
Table 2
IC50 values of 10 treated by CAP in DMSO solution.
IC50
(m
M)
95 % Confidence Intervals (
mM)
Hill Slopes
10 Control (2nd repeat)
CAP 30 s treated 10 (In DMSO)
CAP 10 min treated 10 (In DMSO)
240.5
215.3
246.0
170.8 to 338.8
169.4 to 273.5
235.4 to 257.1
ꢀ1.408
ꢀ1.139
ꢀ3.385
Table 3
IC50 values of 10 treated by CAP 0e40s in culture medium solution.
IC50 M)
(
m
95 % Confidence Intervals (
mM)
HillSlopes
10 Control (3rd repeat)
10 CAP10s
10 CAP20s
10 CAP30s
10 CAP40s
270.9
54.37
31.05
39.21
37.06
95.65
218.6 to 335.6
47.56 to 62.15
26.71 to 36.10
31.27 to 49.16
31.18 to 44.04
79.08 to 115.7
ꢀ0.9283
ꢀ2.228
ꢀ1.764
ꢀ1.765
ꢀ2.012
ꢀ1.667
10 CAP40s þ NAC
times, respectively (Fig. 2 and Table 1). The pyrazolopyrimidinone
derivatives were then determined to be activated within media
solution by CAP-generated ROS in culture media, which can be used
to develop a prodrug strategy combined with CAP treatment. The
CAP treatment can be readily targeted on the tumour site and
locally activate the prodrug, thus minimising non-tumour toxicity
and inducing specific therapeutic effects, especially when prodrugs
have low toxicity in inactive forms. Therefore, compounds 9 and 10
can be potential core scaffold candidates for developing more
sensitive and efficient prodrug derivatives.
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Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
Fig. 5. Two-step synthesis of pyrazolopyrimidinones.
Due to the diversity of CAP commercial devices and prototype
systems, the CAP-induced ROS levels in targeted liquids and gases
can vary. In our previous work, with identical conditions, 30 s of
treatment performed by the same DBD CAP prototype device, tar-
geting culture media, was shown to induce low amounts of
medium to corresponding concentrations, and subsequently
exposed to CAP treatment before being incubated with cells.
Significantly enhanced cytotoxicity were observed with 10 treated
by CAP in culture medium solution (Fig. 4B and C and Table 3). On
the other hand, NAC has been commonly used as ROS scavenger
[64], and we have found that the enhanced cytotoxicity of CAP-
treated 10 in culture medium was significantly inhibited in cul-
ture medium containing NAC (Fig. 4B). Although NAC only partially
recovered the cell viability, this may be due to the instant activation
of 10 when ROS was generated in media or insufficient scavenger
ability of NAC against some specific short-lived reactive species
generated by CAP.
hydrogen peroxide (H2O2) (~20
m mM), and
M), nitrite (NOꢀ2) (~5
nitrate (NOꢀ3) (~30
m
M) within culture media, measured by TiSO4
assay, Griess reagent and 2,6-dimethylphenol assay, which are
significantly less than the IC50 values measured previously for U-
251MG cells (315
mM, >1200 mM and >600 mM respectively)
[55,56]. Although many short-lived reactive species and some long-
lived reactive species were not be able to be characterised, one can
inference that the total amount of CAP-induced reactive species
would be not major, based on the negligible cytotoxicity (Fig. 1C).
On the other hand, glioblastoma cells were demonstrated to have
higher ROS resistance compared to other malignant and non-
malignant cells, which is considered to play major roles in the
therapy resistance of glioblastoma [19,57e59]. In our previous
work, using the same CAP prototype, the IC50 value was determined
to be 4.8 s (95 % confidence range of 4.2e5.6 s) for HeLa cells and
74.26 s (95 % confidence range of 47.24e116.8 s) for U-251MG cells
[19]. Non-malignant cells are usually considered to have lower level
of ROS at natural status, thus their antioxidative capacity is abun-
dant to bear higher levels of ROS [2]. In agreement to this conclu-
sion, using the same system, 50 s of CAP treatment only decreased
around 25 % viability of HEK293 cells in our another work [60].
Although, the cytotoxic effect of 10 is determined to be non-specific
to tumour cells and even is slightly more cytotoxic to HEK293 cells
(Supporting Information Fig. S36), the limited diffusion of reactive
species from the targeted site will limit the exposure of healthy
tissues to the CAP treatment, which will contribute more specific
cancer cells-killing effects, in addition with targeted drug delivery
strategies.
Taken together, the low doses of CAP treatment applied in this
study is sufficient to generate moderate ROS levels in culture media
and cells, which may activate the prodrugs and can be detected by
analytical methods [61] and H2DCFDA assay (Fig. 1A and B),
respectively. The influx of extracellular ROS into the cells is
considered as a main challenge of CAP treatment and is not fully
understood [62]. However, in this study, due to that the cells and
media were treated together between two electrodes of the DBD
CAP system, the intracellular ROS may be generated directly in the
liquid phase of cytoplasm, which can be detected by H2DCFDA [63].
The limited lipid peroxidation-induced cellular responses may also
contribute to the influx of extracellular ROS in this case [61].
As seen in Fig. 4, to distinguish the effects induced by CAP to
cells and compounds, we have investigated the cytotoxicity of CAP-
treated compounds without the direct and immediate effects
caused by CAP treatment to cells. 10 was treated by CAP in DMSO
solution, but demonstrated no significant enhanced cytotoxicity
(Fig. 4A). 100 % DMSO contains no H2O, which impacts on the
generation of ROS [55]. Moreover, DMSO has been demonstrated to
be a scavenger of OH radicals [30], together, this explains the
absence of enhanced cytotoxicity of 10 in a DMSO solution without
substrate. Compound 10 was then first diluted in DMEM culture
Recently, CAP-activated liquids, including water, PBS, saline and
culture media, etc., have been explored for antimicrobial and
anticancer applications [56]. Although reactive species in liquid can
react to generate secondary products post-discharge, such as re-
actions between nitrites and hydrogen peroxide can result in per-
oxynitrite/peroxynitrous acid [65e67], it has been demonstrated
that H2O2, NOꢀ and NOꢀ or other reactive species generated
2
3
depending on the liquids or CAP systems can retain along with their
antimicrobial activities for up to 7 days [55,68] and even longer
term at lower temperatures [67]. In addition, Yan, D. et al. Reported
that short-lived reactive species are removed by overnight storage,
and most of the long-lived reactive species, such as hydrogen per-
oxides in the CAP-activated medium remain [69]. Mohades, S., et al.
also demonstrated that CAP-activated media stored at room tem-
perature for up to 8 h still kills around 50 % cancer cells [70].
Therefore, CAP-activated media stored overnight at 4 ꢁC was
used to test the possible activation effect towards prodrug 10. We
can determine that any oxidised component in the CAP-activated
medium has no effect on the cytotoxicity of prodrug 10 and
short-lived reactive species may make the main contribution to the
activation of the prodrug (Supporting Information Fig. S37b). On
the other hand, CAP has been found to activate various cellular
responses, such as lipid peroxidation [61], accelerated cellular up-
take [21,71], oxidation of protein/DNA/RNA [42], alteration of
intercellular ROS signalling pathways [72], and restoring of cellular
sensitivity to chemotherapy [26]. We incubated CAP-activated U-
251 MG cells with 10, therefore isolating the compounds from ROS,
but observed no significant enhanced cytotoxicity or sensitivity-
restoring effect (Supporting Information Fig. S37c), which demon-
strated that the enhanced cytotoxicity is mainly from CAP-activated
prodrug-like effect rather than CAP-activated cells. In addition, it is
interesting that ROS generator, AAPH, was demonstrated to
decrease 50 % cell viability when combined with low concentra-
tions of 10, compared to 20 % alone, presenting a likely prodrug
activation effect via generation of peroxyl radicals (ROOꢃ) in culture
medium. Moreover, only sufficient generation of peroxyl radicals
would induce enhanced cytotoxicity of 10, comparing incubation
with 3 mM and 0.5 mM of AAPH (Supporting Information Fig. S36).
Although, the use of ROS generator, AAPH, added to understanding
activation mechanism of 10, the potential interaction between
peroxyl radicals and the prodrug-like derivatives, and their effects
at higher concentration, are awaiting further exploration.
In conclusion, in this study, we tested the effect of 15
10

Z. He, C. Charleton, R.W. Devine et al.
European Journal of Medicinal Chemistry 224 (2021) 113736
2641e2645.
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Author contributions
Z.H., P$C., J.S., and J.C. conceived the project and designed the
experiments. Z.H., C$C., R.D., M.K., J.W., G.C., S.G., and J.M. per-
formed the experiments, collected and analysed the data. Z.H., J.S.,
J.C., F.T., B.T., G.K., R.M., D.O., M.D. and W.W. discussed the results
and co-wrote and reviewed the manuscript.
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Funding
[21] Z. He, et al., Cold atmospheric plasma induces ATP-dependent endocytosis of
nanoparticles and synergistic U373MG cancer cell death, Sci. Rep. 8 (2018)
1e11.
This work is supported by Irish Research Council Government of
Ireland Postdoctoral Fellowship Award GOIPD/2020/788 (Z.H., J.C.)
and IRCSET EMBARK grant (G.E.C., J.C.); Science Foundation Ireland
Grant Numbers 14/IA/2626 (P$C., J.C.) and 17/CDA/4653 (B T., P$C.,
J.C.); and by the TU Dublin Fiosraigh Scholarship Programme (S$B.,
J.M., J.C.); Science Foundation Ireland infrastructure grants 16/RI/
3399 and 12/RI/2346/SOF; Maynooth University John Hume
Scholarship (M.K.); Government of Ireland Postgraduate Scholar-
ship from the Irish Research Council (R. D.); Maynooth University
Teaching Studentship (C$C.).
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Biointerphases 10 (2015), 029403.
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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