Antiproliferative chromone derivatives induce K562 cell death through endogenous and exogenous pathways

Article abstract of DOI:10.1080/14756366.2020.1740696

A series of furoxan derivatives of chromone were prepared. The antiproliferative activities were tested against five cancer cell lines HepG2, MCF-7, HCT-116, B16, and K562, and two normal human cell lines L-02 and PBMCs. Among them, compound 15a exhibited the most potent antiproliferative activity. It was also found 15a produced more than 8 μM of NO at the peak time of 45 min by Griess assay. Generally, antiproliferative activity is positively related to NO release to some extent. Further in-depth studies on apoptosis-related mechanisms showed that 15a caused S-phase cell cycle arrest in a concentration-dependent manner and induced apoptosis significantly through mitochondria-related pathways. Human apoptosis protein array assay also demonstrated 15a increased the expression levels of pro-apoptotic Bax, Bad, HtrA2 and Trail R2/DR5. The expression of catalase and cell cycle blocker claspin were similarly up-regulated. In balance, 15a induced K562 cells death through both endogenous and exogenous pathways.

Full text of DOI:10.1080/14756366.2020.1740696

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Antiproliferative chromone derivatives induce
K562 cell death through endogenous and
exogenous pathways
Runwei Jiao, Fanxing Xu, Xiaofang Huang, Haonan Li, Weiwei Liu, Hao Cao,
Linghe Zang, Zhanlin Li, Huiming Hua & Dahong Li
To cite this article: Runwei Jiao, Fanxing Xu, Xiaofang Huang, Haonan Li, Weiwei Liu, Hao Cao,
Linghe Zang, Zhanlin Li, Huiming Hua & Dahong Li (2020) Antiproliferative chromone derivatives
induce K562 cell death through endogenous and exogenous pathways, Journal of Enzyme
Inhibition and Medicinal Chemistry, 35:1, 759-772, DOI: 10.1080/14756366.2020.1740696
To link to this article: https://doi.org/10.1080/14756366.2020.1740696
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 759–772
https://doi.org/10.1080/14756366.2020.1740696
SHORT COMMUNICATION
Antiproliferative chromone derivatives induce K562 cell death through
endogenous and exogenous pathways
Runwei Jiaoa, Fanxing Xub, Xiaofang Huanga, Haonan Lia, Weiwei Liub, Hao Caoc, Linghe Zangc, Zhanlin Lia,
Huiming Huaa and Dahong Lia
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang
b
Pharmaceutical University, Shenyang, P. R. China; Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, P. R. China;
cSchool of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 29 January 2020
Revised 20 February 2020
Accepted 3 March 2020
A series of furoxan derivatives of chromone were prepared. The antiproliferative activities were tested
against five cancer cell lines HepG2, MCF-7, HCT-116, B16, and K562, and two normal human cell lines L-
02 and PBMCs. Among them, compound 15a exhibited the most potent antiproliferative activity. It was
also found 15a produced more than 8 mM of NO at the peak time of 45 min by Griess assay. Generally,
antiproliferative activity is positively related to NO release to some extent. Further in-depth studies on
apoptosis-related mechanisms showed that 15a caused S-phase cell cycle arrest in a concentration-
dependent manner and induced apoptosis significantly through mitochondria-related pathways. Human
apoptosis protein array assay also demonstrated 15a increased the expression levels of pro-apoptotic Bax,
Bad, HtrA2 and Trail R2/DR5. The expression of catalase and cell cycle blocker claspin were similarly up-
regulated. In balance, 15a induced K562 cells death through both endogenous and exogenous pathways.
KEYWORDS
Chromone; nitric oxide;
antiproliferative; selectiv-
ity; apoptosis
GRAPHIC ABSTRACT
1. Introduction
skeletons3–5. It is recognised as a privileged structure and a useful
template for the design of novel compounds with potential
pharmacological interest, particularly in the field of neurodegener-
ative6,7, inflammatory8,9, biocidal10, immune-stimulatory11, infec-
tious diseases12–14, as well as diabetes15 and cancer16–21. With
respect to antitumor activity, chromones demonstrate toxicity
against many kinds of tumour cells, including cervical epithelioid
From ancient times, various natural products have been used as
traditional medicines and are rich sources of bioactive com-
pounds1,2. Chromones (4H-chomen-4-one, 4H-1-benzopyran-4-one)
are widely distributed oxygen-containing natural heterocyclic com-
pounds from the plants of Polygonaceae, Umbelliferae,
Sterculiaceae, Rhamnaceae, Liliaceae, Asteraceae etc., with a ben-
zoylated c-pyrone ring which is a part of the structure of flavonoid carcinoma, breast adenocarcinoma, hepatoma carcinoma, lung
CONTACT Dahong Li
lidahong0203@163.com
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of Traditional
Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China
Supplemental data for this article is available online at here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

760
R. JIAO ET AL.
cancer, leukaemia, colon cancer and so on. The antiproliferative of apoptosis, changes of mitochondrial membrane potential and
mechanisms involve cytotoxicity, anti-metastasis, anti-angiogen-
the expression of apoptosis-related proteins, were also explored.
esis, chemoprevention, immunomodulation and so forth22–27
.
Chromone has emerged as one of the most important syn-
thetic scaffolds for their antitumor activity. Chromone derivative
LY294002 (2-morpholino-8-phenyl-4H-chromen-4-one, Figure 1(A))
is a synthetic protein kinase inhibitor through the blockage of
2. Experimental
2.1. Chemistry
phosphatidylinositol-3-kinase (PI3K) signalling pathway26,28,29
.
All starting materials and solvents were purchased from commer-
cial suppliers and used without purification, unless otherwise
noted. 1H and 13C NMR spectra were measured on Bruker AV400
spectrometers with tetramethylsilane (TMS) as the internal stand-
ard. Chemical shifts were reported in d (ppm). High-resolution
mass spectra were obtained on HClASS XEVOG2XSQTof in the ESI
mode (HR-ESI-MS). All the spectra were in Supplemental data.
Flavopiridol (2-(2-chlorophenyl)-5,7-dihydroxy-8-((3R,4S)-3-hydroxy-
1-methylpiperidin-4-yl)-4H-chromen-4-one, Figure 1(B)) is identified
as the first cyclin-dependent kinase (CDK) inhibitor which blocks
cell cycle progression and induces apoptotic cell death. It has
entered Phase II clinical trials30,31
.
Nitric oxide (NO) acts as an important biological signalling mol-
ecule in a large variety of physiological processes, including
neurotransmission, blood pressure regulation, defence mecha-
nisms, smooth muscle relaxation and tumour growth inhib-
ition32–34. Over the past decade, major advances of NO in cancer
pathogenesis have been witnessed, suggesting an exciting future
in the medical field35,36. Moreover, codelivery of NO with chemo-
therapeutic drugs enhances the suppression of tumour
growth37–40, and it is also discovered to enhance the efficacy of
other treatments, such as photodynamic therapy41–43, radiother-
apy44,45 and ultrasound therapy46,47. However, NO has a half-life
of only a few seconds in an aqueous environment, so NO release
in specific targets is still a crucial challenge for antitumor ther-
apy48,49. A wide range of NO donors have emerged as potential
therapeutics to exploit the biological roles of NO50–55. Some NO
2.1.1. General procedures for the synthesis of compounds 12a–d,
13a–d, 14a–d and 15a–d
A mixture of 10 or 11 (0.5 mmol) and HOBt (0.6 mmol) in anhyd-
rous DMF (5 ml) was stirred at room temperature for 0.5 h. After
the addition of 0.75 mmol 4-(2-aminoethoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole-2-oxide (5a), 4-(3-aminopropoxy)-3-(phenylsul-
fonyl)-1,2,5-oxadiazole-2-oxide (5b), 4-((1-aminopropan-2-yl)oxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (5c) or 3-(phenyl-sul-
fonyl)-4-(2-(piperazin-1-yl)ethoxy)-1,2,5-oxadiazole-2-oxide (5d) and
EDCI (0.75 mmol) to the solution, the mixture was further stirred
at room temperature for 3 h. Then, poured into 20 ml of H2O and
extracted with EtOAc (3 ꢀ 20 ml). The organic layers were com-
donors produce high levels of NO with a wide range of halflives bined, washed with brine, dried over anhydrous Na2SO4 and con-
in vitro and in vivo, and have been widely used in drug research, centrated in vacuo. The crude product was purified by silica gel
especially the type of furoxan (Figure 1(C,D,E))56–58
.
column chromatography eluting with dichloromethane/metha-
nol system.
In this work, 16 chromone derivatives (12a–d, 13a–d, 14a–d
and 15a–d) with NO-releasing furoxan moiety were synthesised
through different linkers. The linkers between the drug and NO
donor influence NO releasing ability56. The antiproliferative activ-
ities against human tumour and normal cells were evaluated.
Furthermore, in-depth apoptosis-related mechanisms of the most
2.1.2 (2-(6-Methyl-4-oxo-4H-chromene-3-carboxamido)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12a)
Yield: 52.6%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.45 (t, 1H,
potent compound 15a, including cell cycle progression, induction J ¼ 6.1 Hz, –NH–), 9.05 (s, 1H, 3-ArH), 8.50 (s, 1H, 20-ArH), 8.06–8.05
Figure 1. The chemical structures of reported chromone and furoxan derivatives. (A,B) Chromone derivatives; (C–E) furoxan-based NO donor derivatives.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
761
(d, 2H, J ¼ 7.2 Hz, 30,50-ArH), 7.80 (s, 1H, 60-ArH), 7.86–7.83 (t, 1H, 137.7, 136.5, 130.4, 128.8, 124.9, 124.7, 120.9, 114.9, 111.1,
J ¼ 7.3 Hz, 40-ArH), 7.77–7.74 (dd, 1H, J ¼ 8.7, 1.9 Hz, 6-ArH),
105.7, 70.6, 56.4, 37.8; HRMS (ESI) m/z calcd for C21H16N3O9S
[MꢁH]ꢁ 486.0607, found 486.0609.
7.72–7.68 (m, 2H, 5,8-ArH), 4.59–4.56 (t, 2H, J ¼ 5.3 Hz, CH2–O),
3.83–3.79 (q, 2H, J ¼ 11.0, 5.6 Hz, CH2–N–), 2.49 (s, 3H, –CH3); 13C
NMR (100 MHz, DMSO-d6) d (ppm): 176.7, 163.3, 163.1, 159.3,
154.4, 137.7, 136.9, 136.8, 136.6, 130.4, 128.9, 125.0, 123.8, 119.0,
115.5, 111.1, 70.5, 37.8, 21.0; HRMS (ESI) m/z calcd for
C21H16N3O8S [MꢁH]ꢁ 470.0658, found 470.0667.
2.1.7 4-(2-((3-Carboxy-6-methoxy-4-oxo-4H-chromen-2-yl)amino)e-
thoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13b)
1
Yield: 35.7%. H NMR (400 MHz, DMSO-d6) d (ppm): 11.74–11.71 (t,
1H, J ¼ 6.6 Hz, –COOH), 8.73–8.57 (q, 1H, –NH–), 8.01–7.96 (m, 1H,
20,60-ArH), 7.85–7.80 (m, 1H, 40-ArH), 7.66–7.61 (m, 2H, 30,50-ArH),
7.41–7.38 (m, 1H, 5-ArH), 7.30–7.28 (m, 2H, 6,8-ArH), 4.69–4.68 (m,
2H, CH2–O), 4.09–4.08 (m, 2H, CH2–N), 3.83 (s, 3H, –OCH3); 13C
NMR (100 MHz, DMSO-d6) d (ppm): 179.8, 177.5, 164.1, 159.1,
156.1, 155.9, 149.0, 137.7, 136.5, 130.4, 128.8, 122.3, 121.2, 118.8,
2.1.3 4-(2-((3-Carboxy-6-methyl-4-oxo-4H-chromen-2-yl)amino)e-
thoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12b)
Yield: 47.6%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.76 (s, 1H,
–COOH), 8.62 (s, 1H, –NH–), 8.00–7.98 (d, 2H, J ¼ 7.6 Hz, 20,60-ArH),
7.85–7.81 (t, 1H, J ¼ 7.5 Hz, 40-ArH), 7.77 (s, 1H, 5-ArH), 7.66–7.62 (t,
2H, J ¼ 8.2 Hz, 30,50-ArH), 7.51–7.48 (dd, 1H, J ¼ 8.4, 1.9 Hz, 6-ArH), 111.1, 107.6, 107.5, 70.1, 56.1, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4;
HRMS (ESI) m/z calcd for C22H18N3O9S [M–H]ꢁ 500.0764,
7.24–7.22 (s, 1H, 8-ArH), 4.69–4.67 (t, 2H, J ¼ 4.6 Hz, CH2-O),
4.08–4.06 (t, 2H, J ¼ 4.5 Hz, CH2–O), 2.38 (s, 3H, –CH3); 13C NMR
found 500.0761.
(100 MHz, DMSO-d6) d (ppm): 180.2, 164.1, 159.2, 153.0, 137.8,
136.6, 135.8, 133.8, 130.4, 128.8, 128.1, 126.0, 125.4, 117.3, 111.6,
96.8, 70.2, 49.2, 20.4; HRMS (ESI) m/z calcd for C22H18N3O8S
2.1.8
4-(3-(6-Methoxy-4-oxo-4H-chromene-3-carboxamido)pro-
[MꢁH]ꢁ 484.0815, found 484.0803.
poxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13c)
Yield: 33.9%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.28–9.25 (t,
1H, J ¼ 5.7 Hz, –NH–), 9.03 (s, 1H, 3-ArH), 8.05–8.04 (d, 2H,
J ¼ 7.5 Hz, 20,60-ArH), 7.90–7.87 (t, 1H, J ¼ 7.5 Hz, 40-ArH), 7.77 (s,
1H, 5-ArH), 7.74–7.73 (m, 2H, 30,50-ArH), 7.51–7.49 (m, 2H, 6, 8-
ArH), 4.50–4.47 (t, 2H, J ¼ 6.0 Hz, CH2–O), 3.89 (s, 3H, –OCH3),
3.51–3.47 (m, 2H, CH2–N), 2.10–2.03 (m, 2H, –CH2–); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 176.4, 162.8, 159.4, 157.7, 150.9,
137.6, 136.6, 130.5, 128.9, 124.9, 124.6, 120.8, 115.2, 111.0, 105.8,
2.1.4 4-(3-(6-Methyl-4-oxo-4H-chromene-3-carboxamido)propoxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12c)
Yield: 48.8%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.27–9.24 (t,
1H, J ¼ 5.8 Hz, –NH–), 9.03 (s, 1H, 3-ArH), 8.06–8.04 (d, 2H,
J ¼ 7.4 Hz, 20,60-ArH), 7.94 (s, 1H, 8-ArH), 7.91–7.87 (t, 1H, J ¼ 7.5 Hz,
40-ArH), 7.77–7.75 (m, 2H, 30,50-ArH), 7.73–7.71 (dd, 1H, J ¼ 6.4,
1.7 Hz, 6-ArH), 7.69–7.67 (d, 1H, J ¼ 8.5 Hz, 5-ArH), 4.49–4.46 (t, 2H,
J ¼ 6.0 Hz, CH2–O), 3.51–3.46 (q, 2H, J ¼ 12.7, 6.48 Hz, –CH2–), 2.46 69.9, 56.3, 35.8, 28.7; HRMS (ESI) m/z calcd for C22H18N3O9S
(s, 3H, –CH3), 2.08–2.03 (m, 2H, CH2–N); 13C NMR (100 MHz, DMSO-
[MꢁH]ꢁ 500.0764, found 500.0782.
d6) d (ppm): 176.7, 163.0, 162.8, 159.4, 154.4, 137.7, 136.7, 136.6,
130.5, 128.9, 125.1, 123.8, 118.9, 115.9, 111.0, 69.9, 35.7, 28.8, 21.0;
HRMS (ESI) m/z calcd for C22H18N3O8S [MꢁH]ꢁ 484.0815,
2.1.9 4-(3-((3-Carboxy-6-methoxy-4-oxo-4H-chromen-2-yl)amino)-
found 484.0803.
propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13d)
Yield: 56.6%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.61 (s, 1H,
–COOH), 8.50 (s, 1H, –NH–), 8.06–8.04 (d, 2H, J ¼ 7.5 Hz, 20,60-ArH),
2.1.5 4-(3-((3-Carboxy-6-methyl-4-oxo-4H-chromen-2-yl)amino)pro-
poxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12d)
7.92–7.89 (t, 1H, J ¼ 7.4 Hz, 40-ArH), 7.78–7.74 (t, 2H, J ¼ 7.8 Hz,
30,50-ArH), 7.35 (s, 1H, 5-ArH), 7.21 (s, 2H, 6,8-ArH), 4.48–4.45 (t, 2H,
J ¼ 5.7 Hz, CH2–O), 3.80 (s, 3H, –OCH3), 3.17–3.16 (d, 2H, J ¼ 2.8 Hz,
CH2–N), 2.18–2.15 (m, 2H, –CH2–); 13C NMR (100 MHz, DMSO-d6) d
(ppm): 159.3, 155.9, 148.9, 137.6, 136.6, 130.5, 129.0, 126.0, 118.8,
111.1, 107.6, 96.4, 69.2, 56.1, 47.8, 29.2; HRMS (ESI) m/z calcd for
Yield: 37.5%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.62 (s, 1H,
–COOH), 8.60–8.43 (m, 1H, –NH–), 8.06–8.04 (d, 2H, J ¼ 7.9 Hz,
20,60-ArH), 7.94–7.88 (m, 1H, 40-ArH), 7.77–7.69 (m, 3H, 30,50,5-ArH),
7.47–7.45 (m, 1H, 8-ArH), 7.21–7.17 (t, 1H, J ¼ 9.6 Hz, 6-ArH),
4.50–4.46 (m, 2H, CH2–O), 3.74–3.69 (q, 2H, J ¼ 12.7, 6.2 Hz, –CH2–),
2.36 (s, 3H, –CH3), 2.20–2.17 (m, 2H, CH2–N); 13C NMR (100 MHz, C25H23N4O9S [MꢁH]ꢁ 555.1186, found 555.1173.
DMSO-d6) d (ppm): 179.8, 163.3, 162.9, 161.4, 159.3, 152.8, 137.6,
136.6, 135.6, 130.5, 129.0, 125.3, 120.5, 117.2, 111.1, 96.4, 69.1,
47.6, 29.2, 20.8; HRMS (ESI) m/z calcd for C25H23N4O8S [MꢁH]ꢁ
2.1.10 2-((1-(6-Methyl-4-oxo-4H-chromene-3-carboxamido)propan-
539.1237, found 539.1229.
2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (14a)
Yield: 28.1%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.43–9.40 (t,
1H, J ¼ 5.5 Hz, –NH–), 9.04–9.03 (d, 1H, J ¼ 6.5 Hz, 3-ArH), 8.07–8.05
(d, 2H, J ¼ 7.4 Hz, 20,60-ArH), 7.97–7.96 (d, 1H, J ¼ 5.6 Hz, 8-ArH),
7.86–7.82 (t, 1H, J ¼ 7.4 Hz, 40-ArH), 7.76–7.74 (dd, 1H, J ¼ 8.7 Hz,
6-ArH), 7.72–7.67 (m, 3H, 30,50, 5-ArH), 5.18–5.14 (m, 1H, –CH–),
3.86–3.80, 3.65–3.59 (m, 2H, –CH2–N–), 2.48 (s, 3H, ArH-CH3),
1.40–1.39 (d, 3H, J ¼ 6.3 Hz, –CH–CH3); 13C NMR (100 MHz, DMSO-
d6) d (ppm): 176.8, 163.3, 163.2, 158.8, 154.4, 137.6, 136.9, 136.5,
130.4, 128.9, 126.0, 125.0, 123.7, 119.0, 115.5, 78.5, 42.6, 21.0, 17.2;
HRMS (ESI) m/z calcd for C21H16N3O9S [M–H]ꢁ 486.0607,
2.1.6 4-(2-(6-Methoxy-4-oxo-4H-chromene-3-carboxamido)ethoxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (13a)
Yield: 45.3%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.48–9.45 (t,
1H, J ¼ 5.7 Hz –NH–), 9.05 (s, 1H, 3-ArH), 8.07–8.05 (d, 2H,
J ¼ 7.5 Hz, 20,60-ArH), 7.86–7.82 (t, 1H, J ¼ 7.5 Hz, 40-ArH), 7.79–7.76
(d, 1H, J ¼ 8.9 Hz, 5-ArH), 7.71–7.67 (t, 2H, J ¼ 8.0 Hz, 30,50-ArH),
7.55–7.53 (dd, 1H, J ¼ 4.4, 2.9 Hz, 6-ArH), 7.51–7.50 (d, 1H,
J ¼ 3.1 Hz, 8-ArH), 4.59–4.56 (t, 2H, J ¼ 5.1 Hz, CH2–O), 3.89 (s, 3H,
–OCH3), 3.84–3.80 (q, 2H, J ¼ 10.7, 5.4 Hz, CH2–N); 13C NMR
(100 MHz, DMSO-d6) d (ppm): 176.4, 163.1, 159.3, 157.8, 150.9, found 486.0609.

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R. JIAO ET AL.
2.1.11 4-((1-((3-Carboxy-6-methyl-4-oxo-4H-chromen-2-yl)amino)- 1H, J ¼ 7.2 Hz, 40-ArH), 7.65–7.63 (t, 2H, J ¼ 7.5 Hz, 30,50-ArH), 7.37
(s, 1H, 5-ArH), 7.28 (s, 2H, 6,8-ArH), 5.28–5.21 (m, 1H, CH–O),
4.06–3.82 (m, 2H, –CH2–), 3.82 (s, 3H, Ar-CH3), 1.41–1.39 (d, 3H,
CH–CH3); 13C NMR (100 MHz, DMSO-d6) d (ppm): 179.9 164.3,
163.2, 158.6, 155.9, 149.0, 137.6, 136.6, 130.3, 128.9, 128.1, 125.9,
122.5, 121.1, 118.9, 111.1, 107.4, 78.0, 70.2, 56.1, 29.5, 16.6; HRMS
propan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (14b)
Yield: 35.6%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.72 (s, 1H,
–COOH), 8.58 (s, 1H, –NH–), 8.00–7.99 (d, 2H, J ¼ 7.2 Hz, 20,60-ArH),
7.84–7.80 (t, 1H, J ¼ 7.2 Hz, 40-ArH), 7.74 (s, 1H, 5-ArH), 7.65–7.61 (t,
2H, J ¼ 7.4 Hz, 30,50-ArH), 7.48–7.47 (d, 1H, J ¼ 7.3 Hz, 8-ArH),
7.21–7.19 (d, 1H, J ¼ 8.9 Hz, 6-ArH), 5.26–5.24 (m, 1H, CH–O),
4.11–4.00 (m, 3H, –CH2–), 2.37 (s, 3H, Ar–CH3), 1.40–1.39 (d, 3H,
CH-CH3); 13C NMR (100 MHz, DMSO-d6) d (ppm): 180.6, 164.2,
163.7, 160.1, 153.6, 138.4, 137.4, 136.4, 134.4, 131.3, 129.8, 126.1,
121.3, 118.0, 111.9, 97.2, 69.9, 48.4, 30.0, 21.6; HRMS (ESI) m/z
calcd for C22H18N3O9S [M–H]ꢁ 500.0764, found 500.0783.
(ESI)
m/z
calcd
for
C22H18N3O10S
[M–H]ꢁ
516.0713,
found 516.0728.
2.1.16 4-(2-(4-(6-Methoxy-4-oxo-4H-chromene-3-carbonyl)pipera-
zin-1-yl)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15c)
1
Yield: 29.9%. H NMR (400 MHz, DMSO-d6) d (ppm): 8.52 (s, 1H, 3-
ArH), 8.04–8.01 (m, 2H, 20,60-ArH), 7.90–7.87 (t, 1H, J ¼ 7.5 Hz, 40-
ArH), 7.77–7.73 (t, 2H, J ¼ 8.1 Hz, 30,50-ArH), 7.69–7.67 (dd, 1H,
J ¼ 7.8, 1.9 Hz, 6-ArH), 7.46–7.43 (m, 2H, 5, 8-ArH), 4.55–4.52 (t, 2H,
J ¼ 10.1 Hz, CH2–O), 3.87 (s, 3H, –OCH3), 3.60–3.57 (t, 2H, J ¼ 4.4 Hz,
–CON–CH2), 3.28–3.26 (t, 2H, J ¼ 7.5 Hz, –CON–CH2), 2.82–2.80 (t,
2H, J ¼ 5.0 Hz, –N–CH2), 2.56–2.54 (t, 2H, J ¼ 5.0 Hz, –CH2–N–C),
2.49–2.47 (t, 2H, J ¼ 4.9 Hz, –CH2–N–C); 13C NMR (100 MHz, DMSO-
d6) d (ppm): 173.5, 162.6, 159.4, 157.3, 156.4, 151.0, 137.8, 136.6,
130.5, 128.7, 124.9, 124.2, 121.9, 120.7, 110.9, 105.4, 69.7, 56.3,
55.9, 53.4, 52.8, 47.1, 41.9; HRMS (ESI) m/z calcd for C22H18N3O10S
[M–H]ꢁ 516.0713, found 516.0714.
2.1.12 4-(2-(4-(6-Methyl-4-oxo-4H-chromene-3-carbonyl)piperazin-
1-yl)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (14c)
Yield: 29.7%. H NMR (400 MHz, DMSO-d6) d (ppm): 8.51 (s, 1H, 3-
1
ArH), 8.03–8.01 (d, 2H, J ¼ 7.4 Hz, 20,60-ArH), 7.90–7.86 (t, 2H,
J ¼ 7.4 Hz, 40,6-ArH), 7.76–7.72 (t, 2H, J ¼ 8.0 Hz, 30,50-ArH),
7.69–7.66 (dd, 1H, J ¼ 8.6, 1.9 Hz, 5-ArH), 7.62–7.60 (d, 1H,
J ¼ 8.6 Hz, 8-ArH), 4.54–4.51 (t, 2H, J ¼ 5.0 Hz, –CH2–O), 3.58–3.56
(t, 2H, J ¼ 4.9 Hz, CON–CH2), 3.27–3.24 (t, 2H, J ¼ 5.4 Hz, CON–CH2),
2.81–2.79 (t, 2H, J ¼ 5.1 Hz, –N–CH2–), 2.55–2.53 (t, 2H, J ¼ 5.2 Hz,
–CH2–N–C), 2.48–2.45 (t, 2H, J ¼ 5.0 Hz, –CH2–N–C), 2.44 (s, 3H,
–CH3); 13C NMR (100 MHz, DMSO-d6) d (ppm): 173.8, 162.5, 159.4,
156.5, 154.5, 137,8, 136.6, 136.2, 136.1, 130.5, 128.7, 127.8, 125.0,
123.8, 122.6, 118.9, 69.7, 55.9, 53.4, 52.8, 47.1, 41.9, 20.9; HRMS
(ESI) m/z calcd for C22H18N3O9S [M–H]ꢁ 500.0764, found 500.0776.
2.1.17 4-(2-(4-(3-Carboxy-6-methoxy-4-oxo-4H-chromen-2-yl)piper-
azin-1-yl)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15d)
Yield: 37.8%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 13.9 (s, 1H,
-COOH), 8.04 (m, 2H, 20,60-ArH), 7.92–7.87 (m, 2H, 40,5-ArH),
7.79–7.75 (m, 3H, 30,50,8-ArH), 7.03–7.00 (dd, 1H, J ¼ 8.9, 3.0 Hz, 6-
ArH), 4.56–4.54 (t, 2H, J ¼ 5.0 Hz, CH2–O), 3.75 (s, 3H, –OCH3), 3.55
(s, 4H, Ar–N–(CH2)2), 2.86–2.84 (t, 2H, J ¼ 5.0 Hz, –N–CH2),
2.62–2.60 (t, 4H, J ¼ 4.6 Hz, –N–(CH2)2); 13C NMR (100 MHz, DMSO-
d6) d (ppm): 190.8, 157.2, 156.9, 154.6, 151.5, 137.8, 136.6, 130.6,
128.7, 126.0, 121.6, 120.6, 120.4, 118.6, 113.0, 105.2, 89.8, 69.9,
60.4, 56.4, 56.2, 55.7, 29.5; HRMS (ESI) m/z calcd for C25H23N4O10S
[M–H]ꢁ 571.1135, found 571.1122.
2.1.13 4-(2-(4-(3-Carboxy-6-methyl-4-oxo-4H-chromen-2-yl)pipera-
zin-1-yl)ethoxy)-3-(phenlsulfonyl)-1,2,5-oxadiazole 2-oxide (14d)
Yield: 27.6%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 14.1 (s, 1H,
–COOH), 8.03 (m, 2H, 20,60-ArH), 7.92–7.89 (m, 1H, 40-ArH),
7.86–7.83 (d, 1H, J ¼ 9.0 Hz, 5-ArH), 7.79–7.75 (m, 3H, 30,50,8-ArH),
7.19–7.17 (dd, 1H, J ¼ 8.3 Hz, 6-ArH), 4.56–4.54 (t, 1H, J ¼ 5.1 Hz,
CH–O), 3.54–3.52 (t, 3H, J ¼ 4.6 Hz, CH–O, Ar–N–CH2), 3.17 (s, 2H,
N–CH2), 2.86–2.84 (t, 2H, J ¼ 5.2 Hz, N-CH2), 2.62–2.60 (t, 4H,
J ¼ 4.1 Hz, –CH2–N–CH2–), 2.25 (s, 3H, –CH3); 13C NMR (100 MHz,
DMSO-d6) d (ppm): 191.2, 160.7, 154.3, 137.8, 136.6, 135.1, 130.6,
129.2, 128.7, 128.1, 127.8, 126.0, 120.2, 117.7, 111.0, 89.8, 70.3,
69.9, 55.7, 53.5, 49.1, 29.5, 20.6; HRMS (ESI) m/z calcd for
C25H23N4O9S [M–H]ꢁ 555.1186, found 555.1171.
2.2. MTT assay
The colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) assay was used to measure the cell viability of
all of the above cell lines following treatment with target hybrids.
Exponentially growing cells were added into 96-well plates at a
concentration of 2000–4000 cells per well. Following attachment
the cells were treated with varying concentrations (64, 16, 4, 1,
0.25, 0.0625 and 0.015625 mM) of target compounds in media and
were kept for incubation for 72 h. MTT (20 mL, 5 mg/mL in PBS)
was added to each well and the cells were incubated for another
3 h at 37 ꢂC. Then the medium was removed, followed by the add-
ition of 150 mL DMSO for each well. After that, the absorbance
(OD) data of each well at 570 nm wavelength were measured by a
Microplate Reader (BIO-RAD), and half inhibition rates (IC50) were
2.1.14
4-((1-(6-Methoxy-4-oxo-4H-chromene-3-carboxamido)pro-
pan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15a)
Yield: 29.5%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 9.45–9.42 (t,
1H, J ¼ 11.6 Hz, –NH–), 9.04 (s, 1H, 3-ArH), 8.07–8.06 (d, 2H,
J ¼ 7.4 Hz, 20,60-ArH), 7.86–7.82 (t, 1H, J ¼ 7.5 Hz, 40-ArH), 7.79–7.77
(m, 1H, 5-ArH), 7.71–7.67 (t, 2H, J ¼ 8.0 Hz, 30,50-ArH), 7.54–7.52 (q,
2H, J ¼ 7.8, 3.2 Hz, 6,8-ArH), 5.18–5.14 (m, 1H, CH–O), 4.54–4.52 (m,
1H, –CH–N–), 3.90 (s, 3H, Ar–CH3), 3.66–3.59 (m, 1H, –CH–N–),
1.41–1.40 (d, 3H, CH–CH3); 13C NMR (100 MHz, DMSO-d6) d (ppm):
176.5, 163.2, 163.1, 158.8, 157.8, 150.9, 137.6, 136.5, 130.4, 128.9,
124.8, 120.9, 114.9, 111.1, 105.6, 78.5, 56.4, 42.6, 17.2; HRMS (ESI)
m/z calcd for C21H16N3O10S [M–H]ꢁ 502.0556, found 502.0544.
calculated59,60
.
2.3. No releasing test
The levels of NO produced by each compound were determined
2.1.15 4-((1-((3-Carboxy-6-methoxy-4-oxo-4H-chromen-2-yl)amino)-
propan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (15b)
by colorimetric assay using
a nitrite colorimetric assay kit
(Beyotime, China) according to the manufacturer’s instructions.
Yield: 22.1%. 1H NMR (400 MHz, DMSO-d6) d (ppm): 11.7 (s, 1H, Incubated in phosphate buffer (pH 7.4) containing 2% dimethyl
–COOH), 8.65 (s, 1H, –NH–), 8.00 (m, 2H, 20,60-ArH), 7.84–7.81 (t, sulfoxide and 10ꢁ5 M test compound (1 ml of 0.2 mM solution in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
763
0.1 M phosphate buffer, pH 7.4) with freshly prepared L-cysteine ethanol for 2 h (or overnight), washed the fixative with PBS, added
(1 ml of 3.6 mM solution in 0.1 M phosphate buffer, pH 7.4) at 100 mL RNase A in 37 ꢂC water bath for 30 min, and finally 400 mL
37 ꢂC for 1, 2 and 3 h without air. After exposure to air for 10 min of PI stain was added, after 30 min under the dark at 4 ꢂC, the
at 25 ꢂC, aliquots of Griess reagent I (50 mL) and Griess reagent II cytometry was used to record the red fluorescence at the excita-
(50 mL) were added to an equal volume (50 mL) of the incubation tion wavelength of 488 nm, and the cell cycle distribution of the
solution of each test compound. After 10 min, the absorbance was DNA content was evaluated.
measured at 540 nm. The nitrite absorbance versus concentration
curve was prepared using 1 M sodium nitrite solution under the
same experimental conditions. The concentration of NO formed
2.6. Hoechst 333258 staining
by a single test compound was calculated using different concen-
trations of nitrite as a standard.
K562 cells were incubated with different concentrations (0, 0.8, 1.6
and 3.2 mM) of 15a for a period of time, the medium containing
the compound was removed, the cell smear was naturally dried,
and immersed in 4% paraformaldehyde for 30 min or overnight to
improve cell permeability, then soaked in PBS, washed for three
2.4. Stability of 15a
Compound 15a was dissolved in culture medium to a final con- times, added appropriate amount of Hoechst 33258 staining solu-
centration of 100 mM from 50 mM stock solution in DMSO. The sol- tion, fully covered, allowed to stand at room temperature for
utions were incubated at 37 ꢂC. An aliquot (20 mL) of the 10 min, finally immersed in PBS again, washed three times and
incubation mixture was taken at different time points (0, 1, 2, 3, 4, sealed. Anti-fluorescence quenching after the liquid was sealed,
5, 6, 8, and 12 h). And the components were analysed by HPLC the staining results were observed under
equipped with a C18 reverse phase column (Shimadzu, LC-6 AD) cence microscope.
with the flow rate of 0.5 ml/min methanol-water (60: 40–100: 0)
a
fluores-
and detection at UV 230 nm.
2.7. Analysis of cellular apoptosis
The cells were placed in six-well plates and incubated at 37 ꢂC for
24 h, after which K562 cells were treated with different concentra-
2.5. Cell cycle study
The cells in the logarithmic growth phase were digested into six- tions (0, 0.8, 1.6, 3.2 mM) of 15a for 24 h, washed with PBS, centri-
well plates. The next day, cells were treated with different concen- fuged, and collected. 500 mL of binding buffer suspension cells,
trations (0, 0.8, 1.6, 3.2 mM) of compound 15a. After 72 h, 5 ꢀ 105 5 mL of Annexin V-FITC, and 5 mL of PI were mixed incubated at
cells were collected and fixed with a volume fraction of 70% room temperature, protected from light, and reacted for 5–15 min,
Scheme 1. Synthesis of 5a–d, 12a–d, 13a–d, 14a–b and 15a–b. Reagents and conditions: (I) ClCH2COOH, NaOH (aq), reflux, 2 h; (II) 30% H2O2, AcOH, rt, 3 h; (III) fum-
ing HNO3, 90 ꢂC, 4 h; (IV) aminoalcohol, THF, NaH, 0 ꢂC, 4 h; (V) POCl3, DMF, ꢁ10 ꢂC, 15 h; (VI) H3NSO3, NaClO2, 0 ꢂC, 12 h; (VII) 5a–d, HOBt, EDCI, DMF, rt, 3 h.

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R. JIAO ET AL.
Table 1. The antiproliferative effects of the target compounds against different human cancer and normal cell lines.
IC50 (lM)a
Compound
HepG2
MCF-7
HCT-116
B16
K562
L-02
PBMC
10
11
5a
5b
5c
5d
>50
>50
21.76 1.35
17.82 1.43
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
29.86 1.47
18.93 1.67
>50
>50
>50
27.94 1.23
22.85 1.46
>50
>50
>50
18.86 0.73
20.34 1.88
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12a
13a
14a
15a
12b
13b
14b
15b
12c
13c
14c
15c
12d
13d
14d
15d
5-Fu
10.59 0.72
19.89 1.31
4.86 0.39
21.34 1.58
22.56 1.39
>50
>50
21.47 1.23
>50
5.67 0.42
25.64 1.57
7.91 0.40
>50
5.38 0.19
9.56 2.37
32.57 1.98
22.14 1.38
>50
13.71 0.33
26.59 1.60
24.83 1.28
>50
13.19 0.53
29.57 1.48
6.74 0.90
26.87 1.61
26.92 1.40
>50
>50
25.36 1.76
>50
7.93 0.44
22.39 1.72
8.64 0.58
>50
7.87 0.32
12.86 1.02
6.86 0.37
16.84 0.74
27.94 1.23
9.72 0.52
31.44 1.34
26.82 1.22
>50
>50
26.87 1.08
>50
10.56 0.53
23.54 1.61
9.96 0.67
>50
11.73 0.52
35.02 1.25
12.62 1.06
5.89 0.26
13.56 0.85
1.61 0.18
12.46 1.03
13.55 0.76
>50
>50
12.46 0.77
>50
2.98 0.14
8.93 0.36
4.94 0.25
>50
2.75 0.17
4.07 0.39
3.94 0.17
36.87 0.62
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.84 1.46
37.59 2.24
15.92 0.66
>50
13.85 0.49
16.89 1.29
26.65 1.92
41.27 2.58
>50
33.64 2.95
>50
28.90 1.43
>50
>50
aIC50: Half inhibitory concentrations measured by the MTT assay. The values are expressed as average standard deviation of three independent experiments.
then analysed with flow cytometry instrument to detect
cell apoptosis.
2.8. Cell mitochondrial membrane potential assay
K562 cells were cultured for 48 h in six-well plates with different
concentrations of 15a (0, 0.8, 1.6 and 3.2 mM), then washed with
PBS and stained with JC-1 in the dark at room temperature. Flow
cytometry was used to measure the number of cells with col-
lapsed mitochondrial membrane potential.
2.9. Quantitation of cellular proteins involved in apoptosis
The relative expression levels of 35 apoptosis-related proteins were
Figure 2. The amounts of NO released by the target compounds 12a–d, 13a–d,
14a–d, and 15a–d.
evaluated using Human Apoptosis Array kit (R&D Systems,
Abingdon, UK) in K562 cells. Proteins were extracted according to
the manufacturer’s protocol from cells treated for 24h with com-
pound 15a (2mM). The tool is fast, sensitive and economical, with
2.0 ml of array buffer added to each well, followed by array, The
capture antibodies are retained in their specific locations, then incu-
bated, transferred, diluted, added to Streptavidin-HRP, shaken. The
platform shaker was incubated on the plate for 30 min, then the
membrane was removed, 1 ml of the prepared Chemi Reagent Mix
was evenly pipetted onto each membrane, incubated, the excess
Chemi Reagent Mix was removed, and the membrane was placed
in a self-developing film cartridge, exposed to X-ray film for
1–10min. The positive signals seen on developed film can be
quickly identified by placing the transparency overlay template on
the array image and aligning it with the pairs of reference spots in
three corners of each array. Creating templates, exporting files,
averaging signals, finding backgrounds, comparing corresponding
signals on the array to determine relative changes in apoptosis-
related protein levels.
3. Results and discussion
3.1. Chemistry
The synthetic routine of target compounds is illustrated in
Scheme 1. Compound
5 was synthesised in a three-step
sequence according to the literature61, and then converted to
various monophenylsulfonylfuroxans (5a–d) by the treatment with
corresponding amino-substituted alcohol, ethanolamine, 3-amino-
propanol, 1-aminopropan-2-ol and N-(2-hydroxyethyl)piperazine
(Scheme 1).
Compounds 8 and 9 were synthesised from 2-hydroxy-5-
methoxyacetophenone (6) and 2-hydroxy-5-methylacetophenone
(7), respectively, by using the Vilsmeier Haack reagent (POCl3 and
DMF) at ꢁ10 ꢂC. Then the aldehyde group was oxidised to carbox-
ylic acid by sulphamic acid and sodium chlorite62. Finally, the furo-
xan intermediates (5a–d) were reacted with the derivatives (10
and 11) of chromone to give the target compounds 12a–d plus

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
765
Figure 3. HPLC of (A) 11 and 15a in MeOH; (B) 15a in cell-free culture medium (cRPMI-1640) after incubation for 1, 3, 6, and 12 h.
Figure 4. Cell cycle analysis of 15a (0, 0.8, 1.6, and 3.2 lM) in K562 cells, cells were stained with PI and then cell cycle distribution was analysed by flow cytometry.

766
R. JIAO ET AL.
Figure 5. Hoechst staining of 15a-treated K562 cells. The red arrows point to the cells with obvious morphological changes of apoptosis.
13a–d and 14a–d plus 15a–d, respectively (Scheme 1). The struc- more potent antiproliferative activities against HepG2, HCT-116 and
tures of all the derivatives were confirmed by 1H NMR, 13C NMR
K562 cells than corresponding ones with methyl group. The results
were in accord with previous literatures that bulky methoxy group
was preferred for antiproliferative activity67,68. In addition, the pres-
ence of carboxyl groups also enhanced antiproliferaitve activity,
which were more easily salified to increase water solubility.
Furthermore, 12d and 14d with a nitrogen-containing heterocyclic
ring showed stronger antiproliferative activities. Other compounds
generally followed the above rules. The target derivatives were sen-
sitive to K562 cell line, of which 15a displayed the most potent
antiproliferative activity with an IC50 value of 1.61mM.
In addition, all the target compounds exhibited weak antiproli-
ferative activities with IC50 values greater than 50 mM against
PBMCs and above 28.90 mM against L-02 cells, which showed
good selectivity between tumour and normal cells.
and high resolution mass spectrum (HR-MS).
3.2. Biological evaluation
3.2.1. Antiproliferative activity
Target chromone/furoxan hybrids (12a-d, 13a-d, 14a-d and 15a-
d) were evaluated for their inhibitory effects against five different
human cancer cell lines (hepatoma HepG2, breast carcinoma MCF-
7, colorectal carcinoma HCT-116, melanoma B16 and chronic mye-
loid leukaemia K562), with the reference fluorouracil (5-Fu)63–66
.
Meanwhile, the activities against the human normal hepatic L-02
cell line and peripheral blood mononuclear cells (PBMCs) were
also evaluated since a potential anticancer drug candidate would
be better to show selective cytotoxicity between malignant and
normal cells.
3.2.2. No releasing ability in vitro
As shown in Table 1, most target compounds displayed more
potent inhibitory activities than corresponding chromone parent
compound. For instance, the antiproliferative activities of the furo-
xan hybrids 12d, 14c, 14d and 15a showed IC50 values ranging
from 1.61 to 15.92 mM against five cancer cell lines. Especially, they
were more sensitive to K562 (IC50 1.61–4.94 mM) and HepG2 (IC50
4.86–7.91 mM) cells. Among 12a, 13a, 14a and 15a, 14a and 15a
The levels of NO released were tested by Griess assay. As shown
in Figure 2, the amounts were basically consistent with the
potency of antiproliferative activity, such as 13a-b, 14b and 15a.
In addition, the faster the amounts of NO released reached the
peak, the stronger activity exhibited. 15a, which showed the
strongest growth inhibitory activity, produced more than 8 mM of
NO at the peak time of 45 min. Generally, the antiproliferative
with a methoxy group at the 5-position of chromone displayed activity is somehow robust related to the amount of NO released.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
767
Figure 6. K562 cells were treated with 15a, stained with annexin V-FITC/PI and analysed by flow cytometry.
3.2.3. Stability of 15a
nucleus was the most significant one. Hoechst 33258, which stains
the cell nucleus, is a membrane permeable dye with blue fluores-
cence. Live cells with uniformly light blue nuclei can be observed
under fluorescence microscope after the treatment with Hoechst
33258. Apoptotic cells have bright blue nuclei on account of kar-
yopyknosis and chromatin condensation; whereas, the nuclei of
dead cells cannot be stained72.
To test that if 15a was easy to hydrolyse, its stability was studied
in cRPMI-1640 culture media supplemented with FBS under cell-
free conditions. The results of HPLC analysis were summarised in
Figure 3. It was observed that 15a was relatively stable in cRPMI-
1640 culture media within 12 h.
In Figure 5, control cells showed no obvious morphological
changes, while K562 cells exposed to 0.8 and 1.6 mM of 15a exhib-
ited brightly blue fluorescence and revealed typical apoptotic
morphology. After the treatment of 3.2 mM of 15a, the K562 cell
membranes were ruptured and the nuclei were fragmented. These
results strongly supported the pro-apoptotic effects of 15a.
3.2.4. Cell cycle analysis
Cell cycle refers to the process that is experienced from the end
of cell division to the end of the next cell division. Numerous anti-
cancer molecules employ their impact via blocking cell cycle pro-
gression, inducing apoptosis, or the merged effects of both69–71
.
To verify the causal relation of cell proliferation inhibition and cell
cycle arrest, K562 cells were treated with different concentrations
of 15a (0, 0.8, 1.6 and 3.2 mM). Effects on cell cycle were deter-
mined by flow cytometry after propidium iodide (PI) staining. As
shown in Figure 4, cells in the S phase increased from 33.76% in
the negative control group to 40.45, 46.68 and 50.28% in a con-
centration-dependent manner. These results revealed that 15a
caused S phase arrest of K562 cells in a concentration-depend-
ent manner.
3.2.6. Cell apoptosis assay
NO has been noted in cancer biology that is associated with can-
cer cell apoptosis73. To further verify 15a induced apoptosis in
K562 cells, K562 cells were treated with different concentrations
(the same as the cell cycle test) of 15a for 72 h. Then, cells were
harvested and stained with Annexin V-FITC and propidium iodide
(PI), and the percentages of apoptotic cells were determined by
flow cytometry. As shown in Figure 6, the apoptotic rates of drug-
treated cells were positively correlated with the concentrations. At
concentrations of 0.8, 1.6 and 3.2 mM, the apoptotic rates were
16.28, 20.56 and 51.87%, respectively, compared with 5.82% in the
3.2.5. Morphological analysis by Hoechst 33258 staining
Cell apoptosis shows characteristic morphological changes, includ-
ing cell shrinkage, chromatin condensation, apoptotic body forma- negative control group, which confirmed that 15a induced apop-
tion, cytoskeletal disintegration, etc. Among them, the change of tosis in K562 cells.

768
R. JIAO ET AL.
Figure 7. 15a induced mitochondrial depolarisation in K562 cells.
Figure 8. The effects exerted by 15a on the expression of apoptosis-related proteins using the Human Apoptosis Array kit in K562 cells.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
769
Figure 9. 15a induced K562 cells death through multiple pathways. Bad and Bax participate in endogenous pathways. Trail R2/DR5 induces cell death through
exogenous pathways. HtrA2 kills cells through non-caspase pathway. In addition, catalase promotes apoptosis by inhibiting NF-jB translocation and reactive oxygen
content. Claspin indirectly induces DNA damage to kill cells.
3.2.7. Mitochondria membrane potential analysis
(TNFR), tumour necrosis factor-related apoptosis-inducing ligand
(Trail), and factor associated suicide/ligand (Fas/FasL) pathways.
The death ligand TRAIL binds to the receptor R2/DR5 on the cell
surface and forms death-inducing signalling complex (DISC),
thereby activates caspase 8, which in turn causes subsequent cas-
cade reactions80,81. The results also showed upregulated expres-
sion of Trail R2/DR5, which were the exogenous pathway results.
HtrA2 is a serine protease located in the mitochondria of
eukaryotes. When cells are stimulated, in addition to inducing
apoptosis through the mitochondrial pathway, Omi interacts with
the antiapoptotic protein HAX-1 (HIS-associated protein X-1) via
the caspases-independent pathway, causing HAX-1 degradation,
thereby increasing the sensitivity of cells to apoptotic stimuli and
inducing apoptosis. P53 also induces HtrA2 phosphorylation by
regulating the actin cytoskeleton to counteract cell migration
Mitochondria are the main organelles that produce ATP and play
important roles in the process of apoptosis74–77. We further exam-
ined the mitochondrial membrane potentials of K562 cells treated
with 15a to confirm its pro-apoptotic effects. K562 cells were
treated with different concentrations of 15a for 48 h, then stained
with the dye 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-
caebocyanine (JC-1). The changes of mitochondrial membrane
potentials were observed. As shown in Figure 7, the depolarisation
of mitochondria increased in a concentration-dependent fashion,
indicating that 15a induced apoptosis through mitochondrial
related pathways.
3.2.8. Effects on apoptosis-related protein
induced by Ras, thereby inhibiting tumour metastasis82,83
.
To further investigate the mechanism of action of 15a in K562
cells, the expression of related apoptotic proteins were tested
using the Human Apoptotic Array Kit (Figure 8). The apoptotic
pathways of cells mainly include the mitochondrial pathway, the
endoplasmic reticulum pathway and the death receptor pathway.
The Bcl-2 family is a major regulator of the mitochondrial
apoptotic pathway. Bad and Bax are pro-apoptotic members that
form homodimers with their family members, increasing mito-
chondrial permeability. Caspase-3 is activated by proteolytic proc-
essing of procaspase-3 and activates caspases chain, and finally
Catalase exists in all known animals and regulates the metabolism
of reactive oxygen species in the body in a stable state, studies
have shown that it inhibits NF-jB activation and promotes
apoptosis84,85
.
To ensure orderly progression of cell cycle, there are many
quality control points known as cell cycle checkpoint proteins.
Claspin, as a tumour suppressor, activates checkpoint kinase 1
(Chk1), sensitises cancer cell DNA during the S phase of the cell
cycle, produces stress responses, and induces DNA damage and
apoptosis86–89. The expression of Claspin is increased to achieve
the purpose of inhibiting the production of tumour cells.
causes apoptosis78,79
. From experimental results (Figure 8),
increased expression of Bad and Bax was observed after the treat-
ment with 15a compared to the control.
The above results indicated that 15a induced apoptosis of
The death receptor pathway regulates apoptosis mainly K562 cells by participating in both endogenous and exogenous
through three pathways, including tumour necrosis factor receptor pathways (Figure 9).

770
R. JIAO ET AL.
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4. Conclusion
In conclusion, 16 furoxan-based chromone derivatives were syn-
thesised and tested for their antiproliferative activity. The results
indicated that most of the target compounds exhibited stronger
antiproliferative activity than the parent compound. 15a showed
the most potent activity with an IC50 value of 1.61 mM against
K562 cells. To further investigate its mechanism of action, the
effects on cell cycle, morphological change, mitochondrial mem-
brane potential and apoptosis-related proteins were evaluated.
The results showed that 15a caused S phase arrest in K562 cells
in a concentration-dependent manner. Cells treated with 15a
showed obvious cell membrane rupture and nuclear fragmenta-
tion. The apoptotic rate of the treated cells was positively corre-
lated with the concentration. Human apoptosis protein array assay
also demonstrated 15a increased the expression levels of pro-
apoptotic Bax, Bad, HtrA2, and Trail R2/DR5. The expression of
Catalase and cell cycle blocker Claspin were similarly up-regulated.
So, 15a induced K562 cells death through multiple pathways,
including endogenous and exogenous pathways, as well as non
caspase pathway. In addition, DNA damage indirectly killed cells.
In brief, 15a as an antitumor drug candidate deserves further 15. Keane WF, Brenner BM. The risk of developing end-stage
investigation.
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Disclosure statement
No potential conflict of interest was reported by the author(s).
17. Yousuf I, Arjmand F, Tabassum SL, et al. Mechanistic insights
into a novel chromone-appended Cu(II) anticancer drug
entity: in vitro binding profile with DNA/RNA substrates and
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Funding
This work was financially supported by Career Development
Support Plan for Young and Middle-aged Teachers in Shenyang
Pharmaceutical University.
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