Synthesis and in vitro anticancer activities of selenium N-heterocyclic carbene compounds

Article type

: Research Article

Synthesis and in vitro anticancer activities of selenium

N-heterocyclic carbene compounds

Sheng Huang^a,1, Xinyu Sheng^a,1, Mianli Bian^a, Zhibin Yang^a, Yunlong Lu^a*and Wukun Liu^a*

^aJiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,

School of Pharmacy, School of Medicine & Holistic Integrative Medicine, Nanjing University of

Chinese Medicine, Nanjing, 210023, China

Corresponding Author

^*Corresponding author. Nanjing University of Chinese Medicine, Nanjing, 210023, China

E-mail: yunlonglu12@njucm.edu.cn (Y. L.);

liuwukun0000@hotmail.com, liuwukun0000@njucm.edu.cn (W. L.)

Tel: +86-25-85811357

Author Contributions

¹Sheng Huang and Xinyu Sheng contributed equally to this work.

Abstract

Fourteen novel selenium N-heterocyclic carbene (Se-NHC) compounds derived from

4,5-diarylimidazole were designed, synthesized, and evaluated as antiproliferative agents. Most of

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process, which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1111/CBDD.13900

them were more effective towards A2780 ovarian cancer cells than HepG2 hepatocellular

carcinoma (HCC) cells. Among them, the most active compound 2b was about 4-fold more active

than the positive control ebselen against A2780 cells. In addition, this compound displayed 2-fold

higher cytotoxicity to A2780 cells than to IOSE80 normal ovarian epithelial cells. Further studies

revealed that 2b could induce reactive oxygen species (ROS) production, damage mitochondrial

membrane potential (MMP), block the cells in the G0/G1 phase, and finally promote A2780 cell

apoptosis.

Keywords: N-heterocyclic carbene; selenium compounds; ovarian cancer; antitumor; apoptosis

1. Introduction

Cancer is one of the diseases with the highest mortality rate in the world. Among them, ovarian

cancer is the main cause of death from all gynecological tumors, and chemotherapy is the main

method for the treatment of metastatic ovarian cancer (Kala et al., 2014). However, commonly

used chemotherapeutic drugs are often limited in clinic due to highly non-specific cytotoxicity and

adverse side effects. Meanwhile, intrinsic and acquired drug resistance is also one of the clinical

disadvantages of chemotherapeutics (Siegel, Naishadham, & Jemal, 2013). Therefore, the

development of new chemotherapeutic drugs is urgent (Banerjee & Kaye, 2013).

Selenium (Se), as an essential micronutrient, plays a significant role in human health

(Hosnedlova et al., 2017; Ibrahim, Kerkadi, & Agouni, 2019). In recent years, researchers focused

on the potential therapeutic effects of Se in cancer treatment (An, Wang, et al., 2018). Among

them, more and more organoselenium derivatives were developed and studied for their lower

toxicity compared to inorganic Se compounds (Reich & Hondal, 2016). Several organoselenium

compounds, including ebselen (Se1), ethaselen (Se2), and Se N-heterocyclic carbene (NHC)

compounds (Se3-6) (Figure 1) have been shown to inhibit cancer cell growth in vitro and in vivo

(Engman et al., 1997; Kamal et al., 2019; Lai et al., 2018; Nguyen et al., 2011; Wang et al., 2012;

Yang et al., 2009; Zeng, Deng, Sang, Zhao, & Chen, 2018). It was also reported that they have

synergistic effects in combination with chemotherapy drugs (Ip, Lisk, & Ganther, 1998; Z. Li et al.,

2009). Therefore, organoselenium compounds with different scaffolds have been used as

chemotherapeutic agents attributed to their significant anticancer bioactivity (Sanmartin, Plano, &

Palop, 2008).

O

Se

N

Se

N

Se

O

Se1 (Ebselen)

Se2 (Ethaselen)

O

N

Se

N

Se

Se5

Se3

Se4

Se6

Fig 1. The structure of compounds Se1-Se6.

NHC was widely used in organic or inorganic chemistry for drug design. As σ-donor or

π-acceptor, NHC could easily combine with some metal elements to form stable and

biologically active complexes (Garcia-Garcia, Martinez, Sanjuan, Fernandez-Rodriguez, &

Sanz, 2011; Gautier & Cisnetti, 2012; Liu & Gust, 2013; Mercs & Albrecht, 2010; Teyssot et

al., 2009). Different transition metals including gold (Casini, Sun, & Ott, 2018; Hickey et al.,

2008; Liu, Bensdorf, & Proetto, 2011, 2012; Long et al., 2020; Meyer et al., 2014; Zou, Lum,

Chui, & Che, 2013), silver (Liu, Bensdorf, Hagenbach, et al., 2011), iridium (Gothe, Marzo,

Messori, & Metzler-Nolte, 2015; Y. Li et al., 2015; C. Yang et al., 2016), platinum (Harlepp

et al., 2019; Rothemund et al., 2020; Tham, Babak, & Ang, 2020) and rhodium (Fan, Bian,

Hu, & Liu, 2019) have been designed as metal-based NHC complexes for chemotherapy,

and many of them exhibited highly effective anticancer activities both in vivo and in vitro.

In our previous work, gold(I) and silver(I) NHC complexes derived from

4,5-diarylimidazole showed high inhibitory effects in MCF-7 and MDA-MB 231 breast

cancer as well as HT-29 colon cancer cells (Liu, Bensdorf, Hagenbach, et al., 2011; Liu,

Bensdorf, & Proetto, 2011; Liu et al., 2012). Preliminary structure activity relationships

(SAR) study demonstrated that the -F and -OMe groups at the para-position of aromatic

rings increased the bioactivity of gold(I) NHC complexes in cancer cells. The substituents

at the nitrogen atoms, the oxidation state of the metal, and the anionic counterion on these

complexes also played significant roles in regulating the bioactivity of them.

Recently, we found that both iodo (1,3-diethyl-4,5-bis(4-methoxyphenyl)

imidazole-2-ylidene)

gold(I)

complex

and

chloride

(η²,η²-cycloocta-1,5-diene)

(4,5-bis(4-fluorophenyl)-1,3-diethylimidazol-2-ylidene) rhodium(I) complex showed strong

antiproliferative activity against hepatocellular carcinoma (HCC) cells (Bian et al., 2020;

Fan et al., 2019). The mechanism study showed that these NHC complexes can powerfully

inhibit the activity of thioredoxin reductase (TrxR). The level of intracellular reactive oxygen

species (ROS) was thereby increased, and it damaged the mitochondrial membrane

potential (MMP) and induced cell apoptosis. In vivo anticancer studies furtherly showed

that gold(I) and rhodium(I) NHC complexes can significantly repress tumor growth in a

HepG2 xenograft mouse model and ameliorate liver injury caused by CCl₄in chronic HCC.

In the continuation of our studies, fourteen novel Se-NHC compounds derived from

4,5-diarylimidazole were designed and synthesized, the antitumor activity of these

compounds were subsequently evaluated. The 4,5-diarylimidazole salts were used as raw

material to obtain Se-NHC compounds and antitumor activities of Se-NHC compounds

were tested in HepG2 cells, A2780 cells, and IOSE80 cells by MTT assay. Bioactivity

research revealed that the most promising compound 2b can induce intracellular ROS

accumulation and promote apoptosis in A2780 cells.

2. Experimental sections

2.1. reagents and methods

All reagents used in the synthesis were obtained from Energy Chemical Company (China)

without further purification. The deuterated solvent was obtained from Cambridge Isotope Lab Inc.

(CIL). Purified TrxR (rat liver) and ct-DNA were obtained from Sigma-Aldrich Co.; JC-1 assay kit

was obtained from APExBio (China). Apotosis kit, ROS detection kit and GSH and GSSG Assay

Kit were obtained from Beyotime (China). DNA content kit was obtained from Key Gen Biotech

(Nanjing, China).

HPLC grade solvents and Millipore (> 18.2 MU) double distilled water were used for the

1

preparation of all spectroscopic and biological research solutions. The H NMR and ¹³C NMR

spectra were measured by Bruker Avance III HD 500 MHz nuclear magnetic resonance

instrument; low resolution mass spectrometry was measured by Aglient 1290-6125 single

quadrupole LC/MS spectrometer, ESI ion Source determination; X-ray single crystal structure data

collected by BRUKER Smart APEX II CCD; UV/Vis spectra were measured by Shimadzu UV2400

spectrophotometer. Fluorescent microscopy was performed by a Leica DMi8 fluorescence

microscope. Flow Cytometry was performed by BD Accuri™ C6 Plus and Beckman Gallios.

Microplate detection was performed by Tecan M1000 Pro.

2.2. Synthesis of selenium compounds

2.2.1. The synthesis of 1a-1n.

The synthesis of compounds 1a, 1b, 1e-1i and 1k-1n has been reported previously (Fan et al.,

2019) . The new analogues 1c, 1d and 1j were prepared by the same route, and their data from

¹H NMR spectra were listed below.

3-benzyl-1-ethyl-4,5-bis(4-methoxyphenyl)-1H-imidazol-3-ium bromide 1c: ¹H NMR (500 MHz,

DMSO) δ 9.68 (s, 1H, NCHN), 7.40-7.27 (m, 5H, Ar_PhH), 7.23-7.06 (m, 4H, Ar_PhH), 6.97 (dd, J =

36.9, 8.6 Hz, 4H, Ar_PhH), 5.37 (s, 2H, NCH₂Ar), 4.12 (q, J = 7.2 Hz, 2H, NCH₂CH₃), 3.76 (d, J =

10.2 Hz, 6H, OCH₃), 1.32 (t, J = 7.2 Hz, 3H, NCH₂CH₃).

1

1-ethyl-3-(2-methoxyethyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-3-ium bromide 1d: H NMR

(500 MHz, CDCl₃) δ 10.32 (s, 1H, NCHN), 7.19-7.07 (m, 4H, Ar_PhH), 6.89-6.79 (m, 4H, Ar_PhH),

4.36 (t, J = 5.0 Hz, 2H, NCH₂CH₂), 4.22 (q, J = 7.3 Hz, 2H, NCH₂CH₃), 3.73 (t, J = 3.7 Hz, 8H,

ArOCH_3,NCH₂CH₂), 3.31 (s, 3H, OCH₃), 1.43 (t, J = 7.3 Hz, 3H, NCH₂CH₃).

1-ethyl-4,5-bis(4-fluorophenyl)-3-(3-hydroxypropyl)-1H-imidazol-3-ium bromide 1j: ¹H NMR (500

MHz, DMSO) δ 9.56 (s, 1H, NCHN), 7.51 (ddd, J = 8.5, 5.4, 1.5 Hz, 4H, Ar_PhH), 7.33 (td, J = 8.8,

2.2 Hz, 4H, Ar_PhH), 4.67 (t, J = 5.0 Hz, 1H, -OH), 4.21 – 4.04 (m, 4H, NCH₂CH₂CH₂OH, NCH₂CH₃),

3.39 (dd, J = 11.1, 5.7 Hz, 2H, NCH₂CH₂CH₂OH), 1.84-1.75 (m, 2H, NCH₂CH₂CH₂OH), 1.31 (t, J =

7.3 Hz, 3H, NCH₂CH₃).

2.2.2. General procedure for the synthesis of 2a-2m

1a-1m (0.83 mmol, 1 eq) was dissolved in dry THF (10 mL), KOtBu (1.66 mmol, 2 eq), selenium

powder (1.24 mmol, 1.5 eq) were added, and the solution was stirred under ice bath for 5 hours.

The products (2a-2m) were isolated by column chromatography on silica gel (mobile phase:

PE/EA, 20:1). The samples were eluted with acetonitrile/H₂O mixture at a constant flow rate. The

purity of all Se-NHC compounds is greater than 95% by HPLC analysis (Figure S5).

1

1,3-diethyl-4,5-bis(4-methoxyphenyl)-imidazole-2-selenone 2a: white powder (yield 60%). H

NMR (500 MHz, CDCl₃) δ 7.11 (d, J = 8.6 Hz, 4H, Ar_PhH), 6.84 (d, J = 8.6 Hz, 4H, Ar_PhH), 4.21 (q, J

13

= 7.1 Hz, 4H, NCH₂CH₃), 3.79 (s, 6H, OCH₃), 1.24 (t, J = 6.8 Hz, 6H, NCH₂CH₃). C NMR (126

MHz, CDCl₃) δ 159.90 (Se=C), 152.81, 131.86, 120.37, 114.08 (Ar_PhC), 129.56 (NC=CN), 55.22

(ArOCH₃), 42.50 (NCH₂CH₃), 14.44(NCH₂CH₃). ESI-MS (+) [m/z]: 416.8 [M+H]⁺. Purity: 95.5% (by

HPLC). Retention time t_R= 15.888 min.

1-ethyl-4,5-bis(4-methoxyphenyl)-3-(2,4,6-trimethylbenzyl)-imidazole-2-selenone 2b: white

powder (yield 66.7%). ¹H NMR (500 MHz, CDCl₃) δ 7.07 (d, J = 8.4 Hz, 2H, Ar_PhH), 6.81 (d, J = 8.5

Hz, 2H, Ar_PhH), 6.63 - 6.54 (m, 4H, Ar_PhH), 6.47 (d, J = 8.5 Hz, 2H, Ar_PhH), 5.58 (s, 2H, NCH₂Ar),

4.27 (d, J = 7.1 Hz, 2H, NCH₂CH₃), 3.78 (s, 3H, ArOCH₃), 3.71 (s, 3H, ArOCH₃), 2.17 (s, 3H,

ArCH₃), 1.94 (s, 6H, ArCH₃), 1.28 - 1.25 (m, 3H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 155.40

(Se=C), 159.88, 159.51, 136.88, 136.61, 131.82, 131.63,130.31, 130.25, 129.01, 120.42, 120.24,

113.95, 113.00 (Ar_PhC), 130.19 (NC=CN), 55.18, 55.17(ArOCH₃), 48.16 (NCH₂Ar), 42.83

(NCH₂CH₃), 20.67, 20.33 (ArCH₃), 14.46 (NCH₂CH₃). ESI-MS (+) [m/z]: 441.2 [M-Se]⁺. Purity:

99.2% (by HPLC). Retention time t_R= 11.764 min.

1-benzyl-3-ethyl-4,5-bis(4-methoxyphenyl)-imidazole-2-selenone 2c: white powder (yield

63.1%). ¹H NMR (500 MHz, CDCl₃) δ 7.23 (d, J = 5.0 Hz, 3H, Ar_PhH), 7.16 - 7.05 (m, 4H, Ar_PhH),

6.86 (dd, J = 8.1, 4.9 Hz, 4H, Ar_PhH), 6.72 (d, J = 8.5 Hz, 2H, Ar_PhH), 5.47 (s, 2H, NCH₂Ar), 4.29 (q,

J = 7.0 Hz, 2H, NCH₂CH₃), 3.78 (d, J = 15.7 Hz, 6H, ArOCH₃), 1.30 (t, J = 7.1 Hz, 3H, NCH₂CH₃).

¹³C NMR (126 MHz, CDCl₃) δ 155.20 (Se=C), 159.95, 159.88, 136.65, 132.07, 131.88, 128.31,

127.56, 127.36, 120.25, 120.20, 114.10, 113.81, 130.07(Ar_PhC), 129.73 (NC=CN),55.24, 55.21

(ArOCH₃), 50.66 (NCH₂Ar), 42.98 (NCH₂CH₃), 14.48 (NCH₂CH₃). ESI-MS (+) [m/z]: 339.2 [M-Se]⁺,

479.0 [M+H]⁺. Purity: 96.1% (by HPLC). Retention time t_R= 16.628 min.

1-ethyl-3-(2-methoxyethyl)-4,5-bis(4-methoxyphenyl)-imidazole-2-selenone 2d: white powder

(yield 65%). ¹H NMR (500 MHz, CDCl₃) δ 7.14 (dd, J = 15.6, 8.7 Hz, 4H, Ar_PhH), 6.85 (dd, J = 8.6,

7.7 Hz, 4H, Ar_PhH), 4.33 (t, J = 6.0 Hz, 2H, NCH₂CH₂), 4.23 (q, J = 7.1 Hz, 2H, NCH₂CH₃), 3.85 -

3.75 (m, 8H, ArOCH_3,NCH₂CH₂), 3.23 (s, 3H, OCH₃), 1.26 (t, J = 7.1 Hz, 3H, NCH₂CH₃). ¹³C NMR

(126 MHz, CDCl₃) δ 153.44 (Se=C), 159.91, 159.87, 132.36, 131.93, 120.39, 120.24, 114.08,

113.92 (Ar_PhC), 130.53, 129.37 (NC=CN), 69.69 (CH₂OCH₃), 58.67 (CH₂OCH₃), 55.24, 55.21

(ArOCH₃), 46.37 (NCH₂CH₂), 42.62(NCH₂CH₃), 14.41(NCH₂CH₃). ESI-MS (+) [m/z]: 367.2 [M-Se]⁺,

447.2 [M+H]⁺. Purity: 95.6% (by HPLC). Retention time t_R= 15.870 min.

1

4,5-bis(4-bromophenyl)-1,3-diethyl-imidazole-2-selenone 2e: white powder (yield 86%). H

NMR (500 MHz, CDCl₃) δ 7.54-7.48 (m, 4H, Ar_PhH), 7.11-7.02 (m, 4H, Ar_PhH), 4.24 (q, J = 7.1 Hz,

4H, NCH₂CH₃), 1.27 (t, J = 7.1 Hz, 6H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 155.12 (Se=C),

132.19, 131.94, 126.84, 123.80 (Ar_PhC), 128.89 (NC=CN), 42.72 (NCH₂CH₃), 14.39 (NCH₂CH₃).

ESI-MS (+) [m/z]: 433.0 [M-Se]⁺. Purity: 97.1% (by HPLC). Retention time t_R= 11.501 min.

1

1,3-diethyl-4,5-bis(4-fluorophenyl)-imidazole-2-selenone 2f: white powder (yield 56.2%). H

NMR (500 MHz, CDCl₃) δ 7.22-7.16 (m, 4H, Ar_PhH), 7.07 (dd, J = 11.8, 5.4 Hz, 4H, Ar_PhH), 4.24 (q,

J = 7.1 Hz, 4H, NCH₂CH₃), 1.27 (t, J = 7.1 Hz, 6H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ

154.39 (Se=C), 164.02, 162.03, 132.46, 132.39, 124.05, 124.02, 116.14, 115.97 (Ar_PhC), 129.03

(NC=CN), 42.65 (NCH₂CH₃), 14.38 (NCH₂CH₃). ESI-MS (+) [m/z]: 415.5 [M+Na]⁺, 313.8 [M-Se]⁺.

Purity: 98.0% (by HPLC). Retention time t_R= 10.628 min.

1-ethyl-4,5-bis(4-fluorophenyl)-3-(2,4,6-trimethylbenzyl)-imidazole-2-selenone 2g: white powder

(yield 40.4%). ¹H NMR (500 MHz, CDCl₃) δ 7.17 - 7.08 (m, 2H, Ar_PhH), 7.04 - 6.96 (m, 2H, Ar_PhH),

6.64 (dd, J = 6.9, 3.9 Hz, 4H, Ar_PhH), 6.58 (s, 2H, Ar_PhH), 5.60 (s, 2H, NCH₂Ar), 4.27 (q, J = 7.1 Hz,

2H, NCH₂CH₃), 2.17 (s, 3H, ArCH₃), 1.93 (s, 6H, ArCH₃), 1.27 (t, J = 5.5 Hz, 3H, NCH₂CH₃). ¹³C

NMR (126 MHz, CDCl₃) δ 156.70 (Se=C), 163.95, 163.63, 161.96, 161.64, 137.13, 136.86, 129.94,

129.73, 129.61, 129.08, 123.90, 123.87, 123.85, 123.82, 115.95, 115.78, 114.73, 114.56 (Ar_PhC),

132.40, 132.34, 132.14, 132.07 (NC=CN), 48.27 (NCH₂Ar), 42.96 (NCH₂CH₃), 29.68, 20.65, 20.27

(ArCH₃), 14.39 (NCH₂CH₃). ESI-MS (+) [m/z]: 417.0 [M-Se]⁺. Purity: 95.2% (by HPLC). Retention

time t_R= 18.680 min.

1-benzyl-3-ethyl-4,5-bis(4-fluorophenyl)-imidazole-2-selenone 2h: white powder (yield 99.8%).

¹H NMR (500 MHz, CDCl₃) δ 7.26-7.16 (m, 5H, Ar_PhH), 7.05 (t, J = 8.6 Hz, 4H, Ar_PhH), 6.90 (d, J =

6.7 Hz, 4H, Ar_PhH), 5.48 (s, 2H, NCH₂Ar), 4.30 (d, J = 7.1 Hz, 2H, NCH₂CH₃), 1.31 (t, J = 7.1 Hz,

3H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 156.84 (Se=C), 164.04, 163.98, 162.05, 161.99,

136.27, 129.51, 129.22, 128.40, 127.57, 127.47, 123.91, 123.88, 123.84, 123.81, 116.12, 115.95,

115.76, 115.59 (Ar_PhC), 132.67, 132.60, 132.45, 132.38 (NC=CN), 50.87 (NCH₂Ar), 43.11

(NCH₂CH₃), 14.37 (NCH₂CH₃). ESI-MS (+) [m/z]: 454.9 [M+H]⁺, 476.9 [M+Na]⁺. Purity: 95.8% (by

HPLC). Retention time t_R= 10.923 min.

1-ethyl-4,5-bis(4-fluorophenyl)-3-(2-methoxyethyl)-imidazole-2-selenone 2i: white powder (yield

40.4%). ¹H NMR (500 MHz, CDCl₃) δ 7.21 (ddd, J = 19.3, 8.3, 5.4 Hz, 4H, Ar_PhH), 7.09 - 6.99 (m,

4H, Ar_PhH), 4.31 (t, J = 5.6 Hz, 2H, NCH₂CH₂), 4.24 (q, J = 7.1 Hz, 2H, NCH₂CH₃), 3.81 (t, J = 5.6

13

Hz, 2H, NCH₂CH₂), 3.22 (s, 3H, OCH₃),1.27 (t, J = 7.1 Hz, 3H, NCH₂CH₃). C NMR (126 MHz,

CDCl₃) δ 154.89 (Se=C), 164.01, 163.99, 162.02, 162.00, 133.10, 133.03, 132.53, 132.46, 124.12,

124.10, 123.95, 123.92, 116.09, 115.92, 115.81, 115.64 (Ar_PhC), 130.18, 128.74 (NC=CN), 69.65

(CH₂OCH₃), 58.61 (CH₂OCH₃), 46.77 (NCH₂CH₂), 42.69 (NCH₂CH₃), 14.31 (NCH₂CH₃). ESI-MS

(+) [m/z]: 422.9 [M+H]⁺, 343.0 [M-Se]⁺. Purity: 99.3% (by HPLC). Retention time t_R= 15.602 min.

1-ethyl-4,5-bis(4-fluorophenyl)-3-(3-hydroxypropyl)-imidazole-2-selenone 2j: white powder

(yield 56%). ¹H NMR (500 MHz, CDCl₃) δ 7.24 - 7.15 (m, 4H, Ar_PhH), 7.07 (td, J = 8.5, 1.4 Hz, 4H,

Ar_PhH), 4.46 - 4.41 (m, 2H, NCH₂CH₂CH₂OH), 4.24 (q, J = 7.1 Hz, 2H, NCH₂CH₃), 3.75 (t, J = 7.2

Hz, 1H, -OH), 3.51 (dd, J = 11.3, 6.5 Hz, 2H, NCH₂CH₂CH₂OH), 1.61 (dt, J = 11.4, 5.8 Hz, 2H,

NCH₂CH₂CH₂OH), 1.26 (t, J = 7.1 Hz, 3H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 154.84

(Se=C),164.09, 162.10, 132.50, 132.47, 132.43, 132.40, 123.70, 123.67, 123.65, 123.62, 116.28,

116.23, 116.10, 116.05 (Ar_PhC), 129.70, 129.17 (NC=CN), 57.19 (NCH₂CH₂CH₂OH), 43.60

(NCH₂CH₂CH₂OH), 42.96 (NCH₂CH₃), 32.29 (NCH₂CH₂CH₂OH), 14.36 (NCH₂CH₃). ESI-MS (+)

[m/z]: 423.0 [M+H]⁺, 343.2 [M-Se]⁺. Purity: 98.7% (by HPLC). Retention time t_R= 14.745 min.

1

4,5-di([1,1'-biphenyl]-4-yl)-1,3-diethyl-imidazole-2-selenone 2k: white powder (yield 77.3%). H

NMR (500 MHz, CDCl₃) δ 7.60 (d, J = 8.0 Hz, 8H, Ar_PhH), 7.45 (t, J = 7.5 Hz, 4H, Ar_PhH), 7.38 (t, J

= 7.4 Hz, 2H, Ar_PhH), 7.32 (d, J = 8.1 Hz, 4H, Ar_PhH), 4.33 (q, J = 7.0 Hz, 4H, NCH₂CH₃), 1.34 (t, J

13

= 7.1 Hz, 6H, NCH₂CH₃). C NMR (126 MHz, CDCl₃) δ 154.08 (Se=C), 141.71, 139.86, 130.93,

128.89, 127.83, 127.33, 127.06, 126.99 (Ar_PhC), 129.69 (NC=CN), 42.75 (NCH₂CH₃), 14.53

(NCH₂CH₃). ESI-MS (+) [m/z]: 429.0 [M-Se]⁺. Purity: 99.0% (by HPLC). Retention time t_R=

19.544 min.

1,3-diethyl-4,5-bis(4'-fluoro-[1,1'-biphenyl]-4-yl)-imidazole-2-selenone 2l: white powder (yield

1

46.0%). H NMR (500 MHz, CDCl₃) δ 7.55 (dd, J = 9.1, 5.1 Hz, 8H, Ar_PhH), 7.34 - 7.29 (m, 4H,

Ar_PhH), 7.14 (t, J = 8.6 Hz, 4H, Ar_PhH), 4.32 (q, J = 7.0 Hz, 4H, NCH₂CH₃), 1.33 (t, J = 7.0 Hz, 6H,

NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 154.23 (Se=C), 163.72, 161.75, 140.74, 135.99, 135.97,

130.99, 128.65, 128.58, 127.20, 127.07, 115.92, 115.75 (Ar_PhC), 129.60 (NC=CN), 42.75

(NCH₂CH₃), 14.53 (NCH₂CH₃). ESI-MS (+) [m/z]: 465.2 [M-Se]⁺. Purity: 97.2% (by HPLC).

Retention time t_R= 11.632 min.

1,3-diethyl-4,5-bis(4'-methoxy-[1,1'-biphenyl]-4-yl)-imidazole-2-selenone 2m: white powder

(yield 84.4%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.66 (dd, J = 11.7, 8.5 Hz, 8H, Ar_PhH), 7.46 (d, J =

8.2 Hz, 4H, Ar_PhH), 7.01 (d, J = 8.7 Hz, 4H, Ar_PhH), 4.13 (q, J = 6.9 Hz, 4H, NCH₂CH₃), 3.79 (s, 6H,

OCH₃), 1.16 (t, J = 7.0 Hz, 6H, NCH₂CH₃). ¹³C NMR (126 MHz, CDCl₃) δ 153.82 (Se=C), 159.54,

141.25, 132.33, 130.91, 128.05, 126.79, 126.40, 114.33 (Ar_PhC), 129.73 (NC=CN), 55.36

(ArOCH₃), 42.72 (NCH₂CH₃), 14.55 (NCH₂CH₃). ESI-MS (+) [m/z]: 489.2 [M-Se]⁺. Purity: 97.2%

(by HPLC). Retention time t_R= 11.949 min.

2.2.3. Synthesis of 2n

1,3-diethyl-imidazole-2-selenone 2n: 1n (686 mg, 3.31 mmol) was dissolved in water (20 mL).

Selenium powder (400 mg, 5.06 mmol) along with Na₂CO₃(920 mg, 6.66 mmol) were then added

and the reaction mixture was heated to reflux for 5 h. The reaction mixture was filtered through

celite, washed with distilled water and was extracted using ethyl acetate. A white powder (403 mg,

1

yield 60%) was obtained under vacuum. H NMR (500 MHz, CDCl₃) δ 6.89 (d, J = 2.0 Hz, 2H,

13

CH=CH), 4.18 (qd, J = 7.2, 1.9 Hz, 4H, NCH₂CH₃), 1.39 (td, J = 7.3, 2.0 Hz, 6H, NCH₂CH₃). C

NMR (126 MHz, CDCl₃) δ 154.06 (Se=C), 118.07 (NC=CN), 44.64 (NCH₂CH₃), 14.46 (NCH₂CH₃).

ESI-MS (+) [m/z]: 204.0 [M+H] ⁺, 125.5 [M-Se]⁺. Purity: 98.7% (by HPLC). Retention time t_R=

11.452 min.

2.3. Cell culture

Human hepatocellular carcinoma cell line HepG2 was obtained from the American Type Culture

Collection (ATCC) and maintained in DMEM (Gibco, USA) supplemented with 10% FBS and 1%

penicillin/streptomycin. Human ovarian cancer cell line A2780 and Human immortalized ovarian

epithelial cell line IOSE80 were purchased from iCell Bioscience Inc (Shanghai, China) and were

grown in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS and 1%

penicillin/streptomycin. All cells reached a maximum of 20 passages during these studies.

2.4. Cell viability assay

2a-2n and ebselen were dissolved in DMF to prepare a 20 mM stock solution. Then, the

solutions were diluted with cell culture medium before treatment. Cells in the logarithmic growth

phase were cultured into 96-well plates overnight and then incubated with the 2a-2n and ebselen

at 37 °C and 5% CO₂for 72 hours. The cell viability was detected by the MTT assay. MTT (5

mg/mL in PBS solution) were added to each well. After incubating for four hours, the medium was

removed and 200 μL DMSO were added to dissolve formazan. Finally, the absorbance data of

each well was detected at 490 nm after shaking on the shaker for 10 minutes.

2.5. Isolated TrxR enzyme assay

Different concentrations of 2b (5 μM, 10 μM, 20 μM, 40 μM) and TrxR were successively

dissolved in reaction buffer, DMF was used as a control. 200 μL of the reaction buffer and 25 μL of

NADPH solution were then added to each well. Subsequently, 25 μL of DTNB (20 mM) solution

was dropwise added to each well to start the reaction. After mixing, the absorbance data were

recorded at 405 nm every 10 s for the first six minutes by microplate reader. The calculation

method was according to our previously published methods (Fan et al., 2019).

2.6. Apoptosis assays

A2780 cells were incubated with 0.1% DMF or 2b in different concentrations for 48 h. Then

A2780 cells were stained with Annexin V-FITC/PI regent before detecting the double fluorescent

signal by flow cytometry (BD Accuri™ C6).

2.7. Cell cycle arrest

A2780 cells (6 x 10⁵cells/well) were cultured in 6-well plates for 24 h. Then, the cells were

treated with 2b for another 24 h. DMF group was as a control. Cells were harvested and

processed using the DNA content kit (Nanjing Key Gen Biotech) according to the manufacturer’s

protocol. Stage of the cell cycle was determined by flow cytometry (Beckman Gallios).

2.8. Intracellular ROS measurement

The fluorescent probe DCFH-DA (Beyotime, China) were used to assess the intracellular ROS

generation. A2780 cells (1 × 10⁵) were evenly seeded in 6-well plate and cultured overnight. After

adhering to the wall, cells were treated with DMF or indicated concentrations of 2b and cultured for

another 6 h. Then, cells were rinsed gently with PBS and DCFH-DA ROS probe (Beyotime, China)

was loaded by the kit method. At last, cells were washed twice with PBS before visualizing

intracelluar fluorescence generation by a fluorescent microscopy (Leica DMi8) under 200

×magnification.

For quantitative measurement of ROS production in A2780 and IOSE80 cells, cells were plated

in 96-well plate at a density of 8000 cells per well, incubated for 24 h before treatment with the

indicated concentrations of 2b for 6 h. Then, cells were treated with fluorescent probe DCFH2-DA

by the same method mentioned above and the fluorescence intensity was tested by a microplate

reader (Tecan M1000 Pro).

2.9. Assessment of MMP

MMP (mitochondrial membrane potential) was detected by the JC-1 dye. A2780 cells were

cultured in a 6-well plate and washed by PBS. Then cells were stained with 5 μM JC-1 for 15min in

darkness. Then remained dye was washed by PBS and red/green fluorescence were observed by

a fluorescent microscopy (Leica DMi8).

2.10. Intracellular GSH/GSSG ratio Measurement

After incubating with 2b for 24 h in A2780 cells, GSH and GSSG Assay Kit (Beyotime, China)

was used to measure the ratio of GSH/GSSG.

2.11. UV-Vis spectral analysis

2b was dissolved in DMF in a concentration of 20 mM and ct-DNA was dissolved in PBS buffer

with pH 7.2 in a concentration of 1.152 mM. 2b was diluted 4000 times to obtain a solution in a

concentration of 20 μM. Next, the ct-DNA solution was gradually dropped into 2b solution

mentioned above. The data of absorption spectra were recorded by spectrophotometer.

2.12. Circular dichroism spectral analysis

2b was incubated with ct-DNA (1.0 × 10^-5M) at 37 °C overnight. The data of 2b with different

concentrations (0, 0.05, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0 × 10^-5M) was recorded range from 235 nm to

300 nm by CD spectrometer.

2.13. Stability analysis

1

2b was dissloved in 10% deuteroxide + 90% dimethyl sulfoxide-d₆and then detected H NMR

spectra in 7 days.

2.14. Interaction with GSH and NAC

2b (2 mM) and GSH (2 mM) were dissloved in dimethyl sulfoxide-d₆and then detected ¹H NMR

spectra in 7 days.

2b (2 mM) and NAC (2 mM) were dissloved in 50% acetonitrile-d₃+ 50% dimethyl sulfoxide-d₆

and then detected ¹H NMR spectra in 7 days.

2.15. X-ray crystallographic analysis

The data of 2b and 2l were collected at 193(2) K and 292(2) K on a BRUKER Smart APEX II

CCD area-detector diffractometer, respectively. CCDC 2051718 and 2052871. DOI:

10.5517/ccdc.csd.cc26vzgj and 10.5517/ccdc.csd.cc26x5nz.

3. Results and discussion

3.1. Chemistry

R₁

ii

iv

vi

i

iii

N

R₂

N

R₂

N

CHO

Br

NH

N

O

HO

Br

Se

2a-2j

Br

1a-1j

R₁

iv

v

vi

N

R₂

N

iv

N

R₂

N

Se

2k-2m

Br

1k-1m

N

R₂

N

vii

R₂

N

Br

1n

Se

2n

compounds

R₂

Et

compounds

R₁

R₂

1a, 2a

F

OMe

1h, 2h

1b, 2b

1i, 2i

1j, 2j

OMe

Br

MeO

HO

1c, 2c

1d, 2d

MeO

Et

1k, 2k

1e, 2e

1f, 2f

Et

1l, 2l

F

1m, 2m

F

MeO

1g, 2g

1n, 2n

F

-

Scheme 1. i: thiamine hydrochloride, rt, ii: formamide, 210 °C, reflux, iii: NaH, ethyl bromide, THF,

reflux, iv: R₂Br, CH₃CN, reflux, 85°C, 2 d, v: K₂CO₃, Pd(PPh₃)₄, ethanol/ H₂O/acetone (3:2:3), reflux, 95

°C, 2 d, vi: Se, KOtBu, THF, 0 °C, 5 h, vii: Se, Na₂CO₃, H₂O, 100 °C, reflux, 5 h.

The 4,5-diarylimidazole salts 1a-1n in the Scheme 1 were synthesized according to our

previously published methods (Fan et al., 2019). In general, benzoin condensation

reactions were carried out to obtain diketone derivatives by anisaldehyde analogues, and

then the products were cyclized with formamide to obtain imidazole derivatives. Two-step

substitution reactions of the imidazole rings were then carried out to obtain

4,5-diarylimidazole salts. In addition, biphenyl imidazole compounds were first obtained by

suzuki coupling reaction. Subsequently, the imidazole salt ligands (1a-1n) were dissolved

in tetrahydrofuran (THF) and reacted with elemental Se powder under the alkaline

condition of potassium tert-butoxide (KOtBu) to obtain the corresponding Se-NHC

compounds 2a-2n (Weiss, Reichel, Handke, & Hampel, 1998). The obtained compounds

1

13

were characterized by H NMR, C NMR, and mass spectra (Figure S4 and S6). The

chemical shift value of the carbene H-atoms for 2a-n disappeared compared to

1

4,5-diarylimidazole salts 1a-n in the H NMR spectra. In ¹³C NMR spectra, the chemical

shift value of C=Se for 2a-n was in the range of 150-160 ppm which was increased by

approximately 15-25 ppm compared to the 4,5-diarylimidazole salts 1a-n. In addition,

positive mode ESI mass spectrometry showed that Se-NHC compounds documented a

peak corresponding to the [M-Se]⁺fragment for 2a-2n.

3.2. Crystal Structure Description

Fig 2. The crystal structure of 2b (left) and 2l (right).

To gain insight into the structural information of these compounds, 2b and 2l were

isolated as single crystals and characterized by X-ray diffraction. The molecular structures

of 2b and 2l were depicted in Figure 2 and Table S1-S10. The compound 2b was

crystallized in dichloromethane and n-hexane in a monoclinic P2₁/c space group: a =

11.4140 (5) Å; b = 12.8499 (6) Å; c = 17.9400 (8) Å; α = 90°; β = 95.821 (2)°; γ = 90°; V =

2617.7 (2) Å³; Z = 4 (Table S1). The selected bond lengths and bond angles were given in

Table S3. Meanwhile, the bond angle was different between N(1) and N(2) side chain:

C(12)-C(11)-N(1), 110.0(3) and N(2)-C(13)-C(14), 114.00(19).

The compound 2l was crystallized in dichloromethane and ethanol in a monoclinic P2₁/c

space group: a = 16.710 (3) Å; b = 10.2283 (16) Å; c = 17.708 (3) Å; α = 90°; β = 106.116

(3)°; γ = 90°; V = 2907.6(8) Å³; Z = 4 (Table S6). The bond angle of C(151)-N(11)-C (221)

and C(151)-N(21)-C(241) in 2l were both 124.0(3) which was different from 2b (Table S8).

The bond lengths of C=Se [1.843(3) and 1.842(4)] are higher than that of general C-N, C-C,

and C-O, and are equivalent to the C=Se bond length in known compounds (1.868(3)),

which are 1.84(3) (Nelson et al., 2014; Seliman et al., 2017).

3.3. Reaction with GSH and NAC

The stability of Se-NHC compounds under physiological conditions is crucial for

biological studies. Hence, the reactivity of the Se-NHC compound 2b in the presence of

1

physiologically important thiols like GSH and NAC, were measured by H NMR spectra

1

(Figure S1and S2). The results showed that none of the new peaks appeared in the H

NMR spectra with GSH and NAC during 7 days, indicating that 2b was relatively stable

1

under the environment of physiologically important thiols. The H NMR spectra of 2b in

deuteroxide was tested as a control and no significant changes were observed, indicating

that 2b was relatively stable in deuteroxide (Figure S3). Above all, we confirmed that 2b

could stay stable under physiological conditions.

3.4 In vitro cytotoxic activities of selenium compounds (2a-2n)

Fig 3. Antiproliferative activity of Se-NHC compounds in HepG2 (A) and A2780 (B) cells at a dose of 20

μM. The results are mean ± SD of three independent experiments.

MTT assay is known as a general experiment to validate the cytotoxicity effect of tested

compounds and it is also a classical method for screening potential anticancer agents.

Thus, in vitro cytotoxicity assays were performed by MTT assay to get an insight into the

antitumor activity of the Se-NHC compounds. Initially, we tested the antitumor activity of

Se-NHC compounds 2a-2n at the same concentration (20 μM) in HepG2 HCC cells and

A2780 ovarian cancer cells by MTT assays. As shown in Figure 3A and 3B, all Se-NHC

compounds have no inhibitory effects in HepG2 cell line while several compounds

including 2b, 2e, and 2h show good antiproliferative effects against A2780 cells. These

results indicated that there was a hint of selectivity of these Se-NHC compounds towards

A2780 cells.

Subsequently, we determined the IC₅₀values of 2a-2n in A2780 human ovarian cancer

cells and IOSE80 human normal ovarian epithelial cells. Ebselen was used as a positive

control. As can been seen from Table 1, several compounds (2a, 2b, 2g, 2e, 2h) exhibited

effective antitumor activity. Among them, antitumor activities of 2a, 2b and 2h were up to

2.6-fold (2a, 9.7 μM), 4-fold (2b, 6.4 μM) and 2-fold (2h, 13.0 μM) more active than ebselen

(25.4 μM). Notably, ebselen and most active compounds which exhibited effective activities

against A2780 cells showed weak cytotoxicity in IOSE80 normal cells. Above all, these

compounds performed equivalent or better selectivity towards cancer cells A2780 than

ebselen.

Hence, we have summarized an SAR study for this series of compounds. As shown in

Table 1, biphenyl Se-NHC compounds 2k, 2l and 2m as well as imidazole Se-NHC

compound 2n showed little cytotoxicity against A2780 cells compared with most

4,5-diphenylimidazole Se-NHC compounds. These results suggested that the benzene ring

at the 4 and 5 positions of the imidazole ring was necessary for bioactivity and larger steric

hindrance substituents like biphenyls could reduce the antiproliferative activity. Next,

Se-NHC compounds 2a (9.7 ± 1.41 μM), 2e (21.1 ± 2.02 μM), and 2k (>160 μM) showed

decreasing antiproliferative activitives against A2780 cells indicating that the inhibitory

activity was related to the substituents at the para position of the benzene ring (inhibitory

activity: Ph < Br< OMe). In addition, compounds with an N(2) -aromatic chain [2b (6.4 ±

1.21 μM), 2g (19.1 ± 3.23 μM), 2h (13.0 ± 1.79 μM) except for 2c (>160 μM)] showed the

same or more active inhibitory effects than those with -aliphatic chains. These results

indicated that the aromatic ring on the N side chain played a significant role in anticancer

activities and it may be attributed to the lipophilicity of the compounds.

Table 1. Antiproliferative effects of 2a-2n against A2780 and IOSE80 Cells after 72 h of

Incubation.

IC₅₀(μM) Mean ± SD

Compounds

SI^a

A2780

25.4 ± 2.80

9.7 ± 1.41

6.4 ± 1.21

> 160

IOSE80

55.4 ± 8.45

51.3 ± 4.86

11.0 ± 2.40

> 160

Ebselen

2a

2.18

5.30

2b

2c

1.72

-

2d

2e

> 160

-

21.1 ± 2.02

85.3 ± 8.63

19.1 ± 3.23

13.0 ± 1.79

34.6 ± 1.50

> 160

43.7 ± 3.87

> 160

2.05

2f

-

2g

2h

2i

34.8 ± 3.06

29.4 ± 2.86

43.4 ± 7.40

> 160

1.83

2.26

1.26

2j

-

2k

> 160

2l

> 160

2m

2n

> 160

^aSI, Selectivity index, SI = IC₅₀(IOSE80) / IC₅₀(A2780).

These results indicated that Se-NHC compounds showed selectivity towards A2780 ovarian

cancer cells and these compounds were not sensitive in HepG2 cells. Among them, compound 2b

exhibited the best antitumor activity against A2780 cells. Moreover, all of Se-NHC compounds

demonstrated selectivity towards A2780 human ovarian cancer cells than IOSE80 human normal

ovarian epithelial cells. The SAR studies also provide some ideas for our later design, such as

expanding the aromaticity of the substituents on the N side chain of the imidazole ring or

introducing aliphatic substituents at the para positions of the 4,5-diaryl groups which may improve

the antiproliferative activity of Se-NHC compounds.

3.5. The isolated TrxR inhibition and interaction with ct-DNA

Se compounds have been well documented to show higher selectivity and sensitivity in cancer

cells and exhibited pro-oxidant properties which can be broadly attributed to different targets such

as protein thiols and DNA binding (Gandin, Khalkar, Braude, & Fernandes, 2018). Selenoprotein

such as TrxR has been considered to be a potential target for selenium compounds (He, Ji, Lai, &

Chen, 2015; Y. W. Liang, Zheng, Li, Zheng, & Chen, 2014). Thus, we studied the TrxR inhibitory

effect and DNA binding affinity of 2b. Unexpectedly, 2b showed weak TrxR inhibitory activity (IC₅₀

> 40 μM) on isolated enzymes using the DTNB assay in Figure 4A, indicating that TrxR is not the

potential target of 2b. Similarly, the absorption of DNA was not changed (Figure 4B) and the

configuration of DNA did not change significantly by circular dichroism (CD) assay with an

increase of 2b concentration (Figure 4C), which also indicated that DNA is not the target of these

Se-NHC compounds.

Fig 4. Inhibition to the activity of purified TrxR by 2b (A); UV/Vis spectra (B) and CD spectra (C)

recorded for the reaction of compound 2b with ct-DNA. r_{b =}[2b]/[ct-DNA].

The above negative results revealed that Se-NHC compounds did not directly interact with

selenoprotein or DNA to exert antitumor activity. Previous studies have shown that Se compounds

were able to alter the intracellular redox balance and a slight additional ROS induction would lead

to oxidative stress dependent cell death in tumor microenvironment (Cairns, Harris, & Mak, 2011;

Gorrini, Harris, & Mak, 2013; Khalkar et al., 2018; Selenius et al., 2012). Hence, the mechanisms

related to tumor cell apoptosis caused by elevated ROS have been another direction for us to

study the mode of action of Se-NHC compounds.

3.6. ROS analysis

Maintaining redox homeostasis is necessary for all healthy cells. The balance between

the generation of ROS and their elimination by cellular antioxidant agents plays an

important role in cellular events. In addition, the level of ROS in tumor cells is excessively

increased because of the aberrant proliferation of cancer cells (Gorrini et al., 2013; Sze et

al., 2020). It was reported that the antitumor effect of organoselenium compounds is

related to the level of ROS (An, Zhang, et al., 2018; Deng, Yu, Cao, Zheng, & Chen, 2015;

Dos Santos et al., 2020; Fernandes & Gandin, 2015; Gandin et al., 2018; He et al., 2015; Y.

Liang, Zhou, Deng, & Chen, 2016; Misra, Boylan, Selvam, Spallholz, & Bjornstedt, 2015;

Yang, Deng, Zeng, Hu, & Chen, 2016; Zhao et al., 2017).

To investigate whether the proliferation inhibitory activity of 2b was associated with

ROS production, the level of ROS in 2b-treated A2780 cells was detected by general

methods in literature (Saleh et al., 2015). As shown in Figure 5A, A2780 cells were

incubated with 2b (10 μM, 20 μM, and 40 μM) for 12 h and the ROS production was

analyzed by fluorescence analysis. The ROS level in cells treated with 2b was higher than

control group. These findings suggested that 2b can activate ROS in a dose-dependent

manner in A2780 cells as expected. To demonstrate whether ROS was related to the

selectivity towards A2780 cells, A2780 human ovarian cancer cells and IOSE80 human

normal ovarian epithelial cells were incubated with 2b in different concentrations (2.5 μM, 5

μM, 10 μM, 20 μM and 40 μM) for 6 h and the production of ROS was measured by ROS

assay kit. As shown in Figure 5B, the production of ROS increased in a dose-dependent

manner in A2780 cells while the production of ROS in IOSE80 was not changed

significantly with the concentration increased, even under a high dose (40 μM). This result

suggested that the antitumor activity of 2b may be due to its ability to disrupt redox

homeostasis in cancer cells and induce ROS accumulation, thereby leading to oxidative

stress dependent cell death of cancer cells. Meanwhile, in normal cell IOSE80, 2b

displayed little ability to induce the accumulation of ROS and this may be the reason for its

selectivity towards A2780 cells than IOSE80.

Fig 5. ROS generation after treatment with 2b in different concentrations for 12 h (A, original

magnification, 200 x ); ROS production of IOSE80 and A2780 after treated with 2b for 6 h (B). The ratio

of GSH/GSSG in A2780 cells treated with 2b for 24 h (C).**p < 0.01 and ***p < 0.001 compared with

group DMF.

It is well known that GSH/GSSG ratio was closely related to ROS level and it was

usually selected as an indicator for antioxidant proteins and oxidative stress. (Lu et al.,

2018). Therefore, we examined whether treatment with 2b can affect the GSH/GSSG ratio.

As shown in Figure 5C, 2b can reduce the GSH/GSSG ratio in A2780 cells. The

decreased GSH/GSSG ratio and increased production of ROS indicated that 2b could

disrupt the redox balance and lead to oxidative stress dependent cell death in A2780 cells.

3.7. MMP

It is well known that the major source of ROS comes from mitochondrial metabolism

(Zhang, Li, Han, Liu, & Fang, 2017). The accumulation of ROS in cells can damage DNA

through oxidative stress, destroy MMP and cause mitochondrial dysfunction, which

ultimately leading to apoptosis and cell death (Bian, Fan, Zhao, & Liu, 2019; Dharmaraja,

2017; Huang et al., 2018; Pelicano, Carney, & Huang, 2004). MMP in A2780 cells treated

with 2b was tested by JC-1 kit. As shown in Figure 6, a dose-dependent decrease in MMP

was observed (red fluorescence turning green fluorescence) indicating the normal

physiological MMP was seriously damaged by 2b in 40 μM. This result suggested that 2b

can induce ROS production, damage MMP, and lead to mitochondrial dysfunction.

Fig 6. MMP imaged by JC-1 staining after treatment with 2b for 24 h (original magnification, 200 x ).

3.8. Cell cycle analysis and apoptosis

The high level of ROS can cause oxidative stress on the cells, which may eventually lead

to cell death or apoptosis (Bian et al., 2019). Therefore, we investigated whether the ROS

production induced by 2b in the A2780 cells could lead to apoptosis. Hence, the apoptosis

of A2780 cells treated with 2b was detected by propidium iodide (PI) staining and

FITC-Annexin V staining (Fan et al., 2019). The A2780 cells were incubated with 2b for 48

h and the state of apoptosis was measured by flow cytometry. As shown in Figure 7A, the

apoptosis in 2b-treated A2780 cells was enhanced in a dose-dependent manner.

Subsequently, the cell cycle progression of 2b-treated A2780 cells was studied by PI

staining and the results showed that the cells were arrested at G0/G1 phase after

treatment for 24 h. (Figure 7B). Thus, 2b could block the A2780 cell cycle in the G0/G1

phase, and finally promote cell apoptosis.

Fig 7. Apoptosis of A2780 cells treated with 2b at different concentrations for 48 h (A); effect of 2b on

cell cycle progression (B). **p < 0.01 and ***p < 0.001 compared with group DMF.

In our previous work, NHC complexes containing different transition metals derived from

4,5-diarylimidazole can inhibit the activity of TrxR and C-terminal thiol/selenol was the

active site (Bian et al., 2020; Fan et al., 2019; Liu, Bensdorf, Hagenbach, et al., 2011; Liu,

Bensdorf, & Proetto, 2011; Liu et al., 2012). We herein further introduced Se into NHC

scaffold to synthesize fourteen Se-NHC compounds and evaluated their biological activity.

Among them, compound 2b with 2,4,6-trimethylbenzyl group on the N(2) side showed the

most potent cytotoxicity and provided a hint of selectivity towards A2780 cells. However,

compound 2b showed weak inhibition of the isolated TrxR which did not meet our original

design and this stimulated us to search for the antitumor mechanism of Se-NHC compounds

.

Inspiringly, numbers of studies showed that Se compounds could induce ROS

accumulation and promote cell apoptosis (Khalkar et al., 2018). Consequently, compound

2b was selected for the following studies, and the action mechanism of compound 2b in

A2780 cells involved accumulation of ROS, mitochondrial dysfunction, and induction of

apoptosis.

4. Conclusion

A series of Se-NHC compounds were synthesized, characterized, and biologically investigated.

A preliminary SAR study showed that compound 2b exhibited an effective cytotoxic effect and

selectivity towards A2780 cells. However, the TrxR and DNA were not the potential targets for

Se-NHC compounds and the experiments showed that the cytotoxicity and selectivity were

attributed to apoptosis activated by ROS. Studies demonstrated that 2b mainly induced the

accumulation of ROS, damaged the MMP, blocked the cells in the G0/G1 phase, and finally

promoted A2780 cell apoptosis. All of the results indicated that 2b represents a potential candidate

for the treatment of ovarian cancer.

Appendix. Supplementary date.

The ¹H NMR and ¹³C NMR spectrum of target compounds; the stability of ¹H NMR spectrum for

2b in deuteroxide, GSH and NAC; crystallographic information of 2b and 2l.

Acknowledgments

We thank the financial supports of the National Natural Science Foundation of China

(No.81703337), the Priority Academic Program Development of Jiangsu Higher Education

Institutions (Integration of Chinese and Western Medicine), the Jiangsu Specially-Appointed

Professors program, the Six Talent Peaks Project in Jiangsu Province of China (No. SWYY-069),

the Open Project of Chinese Materia Medica First-Class Discipline of Nanjing University of

Chinese Medicine (No. 2020YLXK010), and Postgraduate Research & Practice Innovation

Program of Jiangsu Province (No. KYCX20_1564, KYCX20_1510, SJCX20_0532). We also thank

for the experimental support from the experiment center for science and technology, Nanjing

University of Chinese Medicine.

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Article Doi

Synthesis and in vitro anticancer activities of selenium N-heterocyclic carbene compounds

DOI: 10.1111/cbdd.13900

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Full text of DOI:10.1111/cbdd.13900

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