Synthesis and antiproliferative activities of thioxoflavonoids on three human cancer cells

Article abstract of DOI:10.1080/14786419.2018.1448812

Two series of fifteen novel thioxoflavonoids 2a-2h and 4a-4g were synthesized from corresponding flavonoids 1a-1h and 3a-3g by reacting with Lawesson’s reagent, respectively. Their in vitro antiproliferative activities were evaluated on a panel of three human cancer cell lines (Hela, HCC1954 and SK-OV-3) using cell counting kit-8 (CCK-8) assay. The results showed that most of the target compounds exhibited moderate to good antiproliferative activities against the three human cancer cell lines. In particular, thioxoflavonoids 2f and 2g showed the strongest antiproliferative activity on all three human cancer cell lines with IC₅₀ values ranging from 3.34 to 4.67?μM, 4f showed the best antiproliferative activity on Hela cells (IC₅₀ 2.30?μM), 2e showed the best antiproliferative activity on HCC1954 cells (IC₅₀ 2.13?μM) and SK-OV-3 cells (IC₅₀ 2.33?μM). The antiproliferative activities may be involved in their antioxidant activity, which can be speculated by their ability to scavenge free radicals and by their capacity of affecting key redox enzymes.

Full text of DOI:10.1080/14786419.2018.1448812

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Synthesis and antiproliferative activities of
thioxoflavonoids on three human cancer cells
Wei Li, Peipei Han, Shuanglian Cai & Qiuan Wang
To cite this article: Wei Li, Peipei Han, Shuanglian Cai & Qiuan Wang (2018): Synthesis and
antiproliferative activities of thioxoflavonoids on three human cancer cells, Natural Product
Research, DOI: 10.1080/14786419.2018.1448812
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Natural Product research, 2018
https://doi.org/10.1080/14786419.2018.1448812
Synthesis and antiproliferative activities of thioxoflavonoids
on three human cancer cells
Wei Li, Peipei Han, Shuanglian Cai and Qiuan Wang
college of chemistry and chemical engineering, hunan university, changsha, china
ABSTRACT
ARTICLE HISTORY
received 2 January 2018
accepted 2 March 2018
Two series of fifteen novel thioxoflavonoids 2a-2h and 4a-4g
were synthesized from corresponding flavonoids 1a-1h and 3a-
3g by reacting with Lawesson’s reagent, respectively. Their in vitro
antiproliferative activities were evaluated on a panel of three human
cancer cell lines (Hela, HCC1954 and SK-OV-3) using cell counting kit-8
(CCK-8) assay. The results showed that most of the target compounds
exhibited moderate to good antiproliferative activities against the
three human cancer cell lines. In particular, thioxoflavonoids 2f and
2g showed the strongest antiproliferative activity on all three human
cancer cell lines with IC50 values ranging from 3.34 to 4.67 μM, 4f
showed the best antiproliferative activity on Hela cells (IC50 2.30 μM),
2e showed the best antiproliferative activity on HCC1954 cells
(IC50 2.13 μM) and SK-OV-3 cells (IC50 2.33 μM). The antiproliferative
activities may be involved in their antioxidant activity, which can
be speculated by their ability to scavenge free radicals and by their
capacity of affecting key redox enzymes.
KEYWORDS
thioxoflavonoids; lawesson’s
reagent; synthesis;
antiproliferative
CONTACT Qiuan Wang
wangqa@hnu.edu.cn
the supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1448812.
© 2018 Informa uK limited, trading as taylor & Francis Group

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W. LI ET AL.
1. Introduction
Flavonoids, a class of polyphenolic natural products, exhibit unique antioxidant, anticho-
linesterase and cytostatic activity in cell membrane environment, which have been exten-
sively researched in disease therapy and prevention (Certo et al. 2017; Singh et al. 2014).
Polymethoxyflavonoids (PMFs) belong to the flavonoid family with more than four methoxy
groups in flavonoid skeleton, they almost exclusively exist in Citrus plants possessing dis-
tinguished anticarcinogenic, antiinflammatory, antioxidant, and antiatherogenic activities
(Itoh et al. 2008; Miyata et al. 2013).
It was well known that oxidation caused by reactive oxygen species (ROS) is a major cause
of cellular aging, mutagenesis and carcinogenesis (Battin and Brumaghim 2009). Sulfur-
containing compounds usually possess good antioxidant activity, which can be speculated
by their ability to scavenge free radicals or by their capacity of affecting key redox enzymes
and chelating metalions (Battin and Brumaghim 2009; Martins et al. 2015). Interestingly,
recent studied have highlighted that containing sulfur small molecules and enzymes played
a key role in a wide range of fundamental biological functions, which intensely attracted
many researchers to develop new thio-compounds in disease therapy and prevention
(Ravishankar et al. 2016; Mughal et al. 2017). However, despite the fact that the replacement
of the carbonyl (C=O) group by a thiocarbonyl (C=S) group has been usually reported as a
feasible strategy applied in cancer therapy and prevention (Martins et al. 2015; Ravishankar
et al. 2015), the potential anticancer effects of sulfur-containing flavonoids derivatives have
been much less reported. In particular, the potential anticancer properties of polymethoxy
4-thiocarbonyl flavonoids remain almost unexplored.
In order to develop and optimize the flavonoid scaffolds into pharmaceutical agents, we
designed and synthesis two series of fifteen novel thioxoflavonoids with various number of
methoxyl and hydroxyl groups as the continuation of our previous study program in the
natural or synthesized flavonoids (Li et al. 2017; Chen et al. 2016) using Lawesson’s reagent.
Then, they were tested in vitro for their antiproliferative acticity against three human cancer
cell lines Hela (cervical carcinoma), HCC1954 (breast cancer) and SK-OV-3 (ovarian cancer).
2. Results and discussion
The synthesis of the novel thioxoflavonoids was performed according to the reaction path-
ways illustrated in Scheme 1.
Tangeretin (1a), nobiletin (1b), apigenin (3a), luteolin (3c) and quercetin (3f) were used
as starting materials. 5-demethyltangeretin (1c), 5-demethylnobiletin (1d), 3-hydroxy-
4′,5,6,7,8-pentamethoxyflavone (1e), 3-hydroxy-3′,4′,5,6,7,8-hexa methoxyflavone(1f),
3,4′,5,6,7,8-hexamethoxyflavone (1g) and 3,3′,4′,5,6,7,8- heptamethoxyflavone (1h) were
synthesized according to the literature procedures (Chu et al. 2004; Cai et al. 2012). 4′,5,7-tri-
methoxyflavone (3b), 3′,5,7-trihydroxy-4′-methoxyflavone (3d), 3′,4′,5,7- tetramethoxyfla-
vone (3e) and 3,4′,7-trimethoxy-4′,5-dyhydroxyflavone (3g) were synthesized according to
the literature procedures (Wu et al. 2012). They were treated with Lawesson’s reagent in
anhydrous toluene, upon replacement of the oxygen atom of the carbonyl moiety by sulfur
atom, to obtain the corresponding 4-thiocabonyl derivative thioxoflavonoids in excellent
1
yield ranging from 65 to 86% yield. All of synthesized compounds were confirmed by H
NMR, 13C NMR, MS and HRMS analytical data. The most difference in NMR spectra between

NATURAL PRODUCT RESEARCH
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W. LI ET AL.
1
thioxoflavonoids and corresponding flavonoids were shown in H-3 and C-4. The H NMR
data showed that the H-3 chemical shifts of thioxoflavonoids signal were observed in 7.30–
7.50 ppm, while the H-3 chemical shifts of corresponding flavonoids were observed in
6.50–6.64 ppm. The 13C NMR data showed that the C-4 chemical shifts of thioxoflavonoids
signal were observed in 190–200 ppm, while the C-4 chemical shifts of corresponding fla-
vonoids were mostly observed in 171–182 ppm. The molecular masses of the synthetic
compounds were confirmed by EI or ESI. Their mass spectra showed the presence of molec-
ular ion peaks, M+H or M−H ion peaks.
All the synthetic thioxoflavonoids and corresponding flavonoids precursor were screened
in vitro for antiproliferative activity, which was assessed using the cell counting kit-8 (CCK-8)
assay with three human cancer cell lines (Hela, HCC1954 and SK-OV-3), and cis-Platin,
Paclitaxel and staurosporin were used as positive controls. Their activities were expressed
by the IC50 value (concentration of drug inhibiting 50% cell growth), which was presented
in Table 1. The compounds having IC50 value more than 100 μM were considered inactive.
As indicated in Table 1, a good number of synthesized target compounds toward these
three cancer cells exhibited moderate to potent antiproliferative activities. Compounds 2d,
3e, 4b and 4e did not displayed any antiproliferative activity to the three cancer cells
Table 1. the antiproliferative activity (Ic50 in μM) of compound 1a-1h, 2a-2h, 3a-3g and 4a-4g on three
human cancer cells.
Compound
Hela
HCC1954
SK-OV-3
1a
42.96
42.97
7.52
>100
>100
21.15
43.45
32.23
22.89
93.28
66.66
30.67
11.42
19.48
>100
2.13
>100
>100
39.02
66.95
51.40
55.52
45.37
57.46
45.35
19.01
36.08
>100
2.33
1b
1c
1d
32.36
>100
35.16
41.57
31.41
40.31
15.64
11.36
>100
16.93
3.69
4.00
25.87
4.52
23.79
4.85
10.65
>100
19.90
17.63
17.90
>100
7.03
>100
>100
2.30
22.12
13.30
0.0055
0.0117
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
4.67
4.56
3.89
3.34
2g
2h
35.33
11.59
>100
11.11
11.27
>100
92.52
18.11
64.15
>100
14.38
32.79
>100
28.54
20.36
29.32
0.009
0.037
49.50
39.89
>100
34.22
>100
>100
87.01
>100
>100
>100
44.88
54.15
>100
28.75
31.97
18.66
0.0028
0.0031
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
cis-Platina
Paclitaxela
staurosporina
acis-Platin and Paclitaxel and staurosporin were employed as positive controls.

NATURAL PRODUCT RESEARCH
5
(IC50 > 100 μM). Compounds 1c, 2c, 2f, 2g, 3a, 3c, 3d, 4c and 4g were more potent against
Hela cells with IC50 values of 2.30–11.36 μΜ than the positive control cis-Platin (one of the
most widely used anticancer drugs) with IC50 13.30 μΜ. Compounds 1c, 1f, 2b, 2c, 2e, 2f,
2g, 3a, 3c, 3d, 3g, 4c, 4f and 4g were more potent aginst HCC1954 cells with IC50 values of
2.13–28.54 μΜ than the positive control cis-Platin (IC50 29.32 μΜ), and compounds 2e, 2f
and 2g were more potent against SK-OV-3 cells with IC50 values of 2.33–3.89 μΜ than the
positive control cis-Platin (IC50 18.66μΜ). In particular, compound 1c, 2f, 2g, 3a, 3c, 4c and
4f on Hela cells, compounds 2e, 2f and 2g on HCC1954 and Sk-OV-3 cells showed markedly
potency with IC50 value below 10μΜ. Compound 1a, 1b and 3b showed selective antipro-
liferative activity against Hela cells.
In all compounds, polymethoxy thioxoflavonoids 2f and 2g showed excellent antiprolif-
erative activity on all three human cancer cell lines with IC50 values ranging from 3.34 to
4.67 μM, 2e showed the best antiproliferative activity on HCC1954 cells (IC50 2.13 μM) and
SK-OV-3 cells (IC50 2.33 μM), polyhydroxy thioxoflavonoid 4f showed the best antiproliferative
activity on Hela cells (IC50 2.30 μM). This suggested that polymethoxy thioxoflavonoids 2e,
2f, 2g and polyhydroxy thioxoflavonoid 4f were promising candidates for further optimisa-
tion and development as antiproliferative agents on the three cancer cells.
The antiproliferative mechanisms of thioxoflavonoids may be involve in their antioxidant
activity, which can be speculated by their ability to scavenge free radicals and chelating
metal ions, or by their capacity of affecting key redox enzymes such as thioredoxin, catalase
and GPx. The free radical scavenging activity of thioxoflavonoids is highly dependent on
their structural features and the electronic contribution of substituents (Battin and
Brumaghim 2009; Martins et al. 2015). Molecular recognition in the target-binding site, such
as thioredoxin, catalase and GPx, in these cancer cells may be the reason for different behav-
ior of these compounds.
3. Experimental
See Supplementary materials.
4. Conclusion
In summary, we have developed a simple, versatile and rapid methodology to synthesize
4-thioxoflavonoids from 4-oxoflavonoids using lawesson’s reagent. Synthesis of thioxofla-
vonoids was carried out with the substitution of variable hydroxyl and methoxyl groups in
the A, B and AB-ring of the respective flavonoids. The reagents offer high functional groups
tolerance, and have no influence on substitution pattern of flavonoids. The antiproliferative
activities in vitro tested results showed that most of the target compounds exhibited mod-
erate to good antiproliferative activities against three hunan cancer cell lines Hela, HCC1954
and SK-OV-3. Replacing the 4-carbonyl moiety with 4-thicarbonyl generally enhanced the
antiproliferative activity. The antiproliferative mechanisms of thioxoflavonoids may be
involve in their antioxidant activity, which can be speculated by their ability to scavenge
free radicals, or by their capacity of affecting key redox enzymes such as thioredoxin, catalase
and GPx. Polymethoxy thioxoflavonoids 2e, 2f, 2g and polyhydroxy thioxoflavonoid 4f were
promising candidates for further optimisation and development as anticancer agents.

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W. LI ET AL.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China [grant numbers
J1210040, 21173074].
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