Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing properties

Article abstract of DOI:10.1016/j.ejmech.2018.04.009

To explore antitumor agents with high efficiency and selectivity, two series of 16 H₂S donating evodiamine derivatives 8–12 were synthesized and characterized by ¹H NMR, ¹³C NMR and HRMS. Their antiproliferative activities

Full text of DOI:10.1016/j.ejmech.2018.04.009

Accepted Manuscript
Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis
inducing properties
Xu Hu, Runwei Jiao, Haonan Li, Xianhua Wang, Haoda Lyu, Xiang Gao, Fanxing Xu,
Zhanlin Li, Huiming Hua, Dahong Li
PII:
S0223-5234(18)30336-2
10.1016/j.ejmech.2018.04.009
EJMECH 10358
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2018
Revised Date: 2 April 2018
Accepted Date: 3 April 2018
Please cite this article as: X. Hu, R. Jiao, H. Li, X. Wang, H. Lyu, X. Gao, F. Xu, Z. Li, H. Hua, D.
Li, Antiproliferative hydrogen sulfide releasing evodiamine derivatives and their apoptosis inducing
properties, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.009.
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Antiproliferative Hydrogen Sulfide Releasing
Evodiamine Derivatives and Their Apoptosis
Inducing Properties
Xu Hua, Runwei Jiaoa, Haonan Lia, Xianhua Wangb, Haoda Lyua, Xiang Gaoa,
Fanxing Xuc, Zhanlin Lia, Huiming Huaa, Dahong Lia,
aKey Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, and School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, PR China
bSchool of Public Health, Qingdao University, 38 Dengzhou Road, Qingdao 266021, PR China
cWuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang
110016, PR China
Corresponding authors. E-mail addresses: lidahong0203@163.com (D. Li).

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ABSTRACT
To explore antitumor agents with high efficiency and selectivity, two series of 16
H2S donating evodiamine derivatives 8-12 were synthesized and characterized by 1H
NMR, 13C NMR and HRMS. Their antiproliferative activities were tested against five
cancer cell lines (Bel-7402, MCF-7, SGC-7901, Caco-2 and HL-60) and human
normal peripheral blood mononuclear cells. Among them, compound 12c showed the
most potent inhibitory activities against human leukemia HL-60 and epithelial
colorectal adenocarcinoma Caco-2 cells with IC50 values of 0.58 and 2.02 µM,
respectively. Additionally, high selectivity was also observed between human normal
peripheral blood mononuclear cells and human leukemia HL-60 cells. Further
mechanism studies confirmed that 12c could induce apoptosis, arrest cell cycle at the
G2/M phase and lead to mitochondrial dysfunction in HL-60 cells. Furthermore,
western blot assay demonstrated that 12c induced the intrinsic apoptotic
mitochondrial pathway by upregulating protein expression of Bax, cytochrome c,
caspase-3, -9 and p53, and downregulating the relative levels of Bcl-2. The levels of
cell cycle related proteins cyclin B1 and cdc2 were also downregulated in which
G2/M phase arrest was confirmed. Overall, 12c possessed immense potential for the
discovery of antitumor candidates with high efficiency and selectivity.
Keywords: natural product, antiproliferative activity, selectivity, apoptosis

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1. Introduction
Evodiamine (1), which is a quinazolinocarboline alkaloid, was first isolated from
the fruits of Euodia rutaecarpa Bentham [1]. It exhibits diverse pharmacological
activities, such as anti-Alzheimer’s disease [2,3], anti-cancer [4], analgesic activities
[5], anti-obesity [6], anti-hyperlipidemia [7] and anti-inflammation [8]. It also plays a
vital role for preventing osteolytic diseases [9], reducing caffeine-induced sleep
disturbances and excitation [10], ameliorating liver fibrosis via TGF-β1/Smad
signaling pathway [11,12], and inhibiting angiotensin -induced cardiomyocyte
hypertrophy [13]. Among them, the potential for evodiamine and its analogues as
potent cancer chemotherapeutic agents has intrigued considerable research for new
analogues with broad-spectrum antitumor activity [14–17], including human
hepatoma [18], urothelial cell carcinoma [19], renal carcinoma [20], lung cancer [21],
nasopharyngeal carcinoma [22], leukemia [23], gastric cancer [24], oral cancer [25],
colorectal cancer [26], pancreatic cancer [27], bladder cancer [28], breast cancer [29],
and ovarian cancer [30]. Moreover, the mechanisms which have been investigated
demonstrated that evodiamine could induce cell growth arrest and apoptosis in human
urothelial cell carcinoma and renal carcinoma cells [19,20]. Treatment of
hepatocellular carcinoma cells with evodiamine exerted antiproliferative activity by
inducing Akt-mediated apoptosis and a WW (two conserved tryptophans)
domain-containing oxidoreductase-dependent pathway [18,31]. Besides, western blot
analysis indicated that evodiamine could mediate the up-regulation of apoptotic
proteins Bax/Bcl-2 and cleaved-caspase 3 in hepatocellular carcinoma cells [18].
Whereafter, some researchers reported the effect of evodiamine on the interaction
between DNA methyltransferases (DNMTs) and target microRNAs during malignant

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transformation of the colon by transforming growth factor-β1 (TGF-β1) [32].
Evodiamine showed broad antitumor activities through various mechanisms. However,
moderate efficacy, as well as the poor selectivity between cancerous and normal cells,
greatly impeded its further preclinical development and clinical applications. In order
to overcome these disadvantages, novel evodiamine derivatives should be explored.
Hydrogen sulfide (H2S) [33], a colorless and water-soluble gas with the smell of
rotten eggs, is now recognized as an important gasotransmitter, after the extensive
study of nitric oxide (NO) and carbon monoxide (CO). H2S is produced endogenously
via enzymatic reactions of cysteine, homocysteine and cystathionine mediated by
cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate
sulfurtransferase (3-MST) [34]. Over the past decade, numerous studies have shown
that H2S plays significant roles in physiology and pathology regarding myocardial
reperfusion injury [35], hypertension [36], atherosclerosis [37], stroke [38],
neuroinflammation [39], diabetes mellitus [40] and so on. Recently, H2S was found to
show anticancer activity [41,42], which could trigger apoptosis via activation of
MAPK and caspase-3, and sulfhydration of NF-κB [43], enhance cell proliferation of
human gastric cancer AGS cells [44], promote autophagy of hepatocellular carcinoma
cells through EGFR/ERK/MMP-2 and PI3K/Akt/mTOR signaling pathways [45,46],
and induce C6 glioma cell growth through activation of the p38
MAPK/ERK1/2-COX-2 pathways [47]. However, H2S is not used directly in clinic
because of uncontrollable dose and high toxicity. Researchers often use H2S releasing
agents (H2S donors) to modulate H2S levels and enable new investigations [48,49]. It
was reported the slow-releasing H2S donor, GYY4137, exhibited potent
anti-hepatocellular carcinoma activity through blocking the signal transducer and
STAT3 pathway [50,51]. Thereafter, H2S-releasing diclofenac derivatives not only

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increased the activity of anti-inflammatory and reduced the side effects of diclofenac,
but also inhibited breast cancer-induced osteoclastogenesis and prevented osteolysis
[52]. Thenceforward, increasing studies have been reported on the antitumor effects
of H2S donors [53–57]. With the development of H2S donating derivatives (Fig. 1),
many H2S donors were found, such as dithiothione derivatives ADTOH [58],
α-thioctic acid [59], thiobenzamide derivatives [60], iminothioethers [61], cyclic acyl
disulfides
and
acyl
selenylsulfides
[62],
tetrasulfides
[63]
and
1,2,4-thiadiazolidin-3,5-diones [64]. In general, substantial progress has been
achieved in the application of H2S donors towards the treatment of human cancers
over the past decade. Therefore, design and synthesis of H2S releasing hybrids is an
effective and promising strategy for cancer treatment.
In this study, we wanted to continue our studies [65,66] of evodiamine to seek
highly selective antiproliferative drug candidates by combining the potent natural
compound with H2S donors. Aforementioned predicament inspired us to synthesize
16 derivatives based on two kinds of exogenous H2S donors: ADTOH and α-thioctic
acid. Resulting hybrids were evaluated against human cancer Bel-7402, MCF-7,
SGC-7901, Caco-2 and HL-60 cell lines, and normal peripheral blood mononuclear
cells (PBMCs) for antiproliferative activities. Additionally, further mechanisms
regarding cell cycle progression, induction of apoptosis, dysfunction of mitochondrial
membrane potential and expression of apoptosis-related and cell cycle-related
proteins by representative derivative 12c were also investigated.
2. Result and Discussion
2.1. Chemistry
The synthetic routine of target compounds is illustrated in Scheme 1 and 2. First, 1

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was
treated
with
corresponding
bromohydrin
(2-bromine
ethanol,
3-bromine-1-propanol or 6-bromine-1-hexanol), bromate ethyl ester (ethyl
bromoacetate, ethyl 4-bromobutyrate, ethyl 5-bromovalerate) in the presence of NaH
and anhydrous N,N-dimethylformamide (DMF) to offer 2a–d and 3a-c in high yields.
Then 3a-c were hydrolyzed by potassium hydroxide to generate the intermediates
4a-c. Next, the substitution reactions of 5 with corresponding dibromo reagents
(dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane) were completed smoothly
under the condition of potassium carbonate and anhydrous acetone (Scheme 1).
Finally, the target H2S donating evodiamine derivatives 8a-d were synthesized
from 2a–d accordingly with α-thioctic acid
7
in the presence of
carbodiimide
4-dimethylaminopyridine/1-ethyl-3-(3-dimethylaminopropyl)
hydrochloride (DMAP/EDCI) in anhydrous dichloromethane (DCM) at room
temperature for 8-12h. The derivatives 9a-c were got from 2a–d by the reaction with
5 under the condition of DMAP/EDCI in anhydrous DCM. Then the H2S donor
intermediates 6a-c were treated with the evodiamine derivatives 4a-c to give the
target compounds 10a-c, 11a-c and 12a-c in the presence of triethylamine (TEA) and
anhydrous DMF (Scheme 2).
2.2. Biological evaluation
2.2.1 Antiproliferative activity
The antiproliferative activities of 16 target hybrids (8a–d, 9a-c, 10a-c, 11a-c and
12a-c) against five different kinds of human cancer cell lines (human hepatoma cell
line Bel-7402, human breast carcinoma cell line MCF-7, human gastric carcinoma
cell line SGC-7901, human epithelial colorectal adenocarcinoma cell line Caco-2 and
human leukemic cell line HL-60) and PBMCs were tested, compared with lead
compound evodiamine (1), ADTOH (5), α-thioctic acid (7) and positive control

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5-fluorouracil (5-FU).
As shown in Table 1, most target compounds were sensitive to Caco-2 and HL-60
cell lines, which displayed more potent inhibitory activities than evodiamine, and
some of them showed superior cytotoxic activities to 5-FU. While, in Bel-7402 and
SGC-7901 cell lines, target compounds showed almost no cytotoxicity. In MCF-7 cell
line, only 8a was stronger than parent compound 1 with IC50 value of 9.47 µM.
Interestingly, 10a-c, 11a-c, 12a-c incorporated with ADTOH showed stronger
activities than 8a-d with α-thioctic acid against HL-60 cells. When R was (CH2)2 (8a),
the antiproliferative activities were stronger than those with (CH2)3, (CH2)3-O-(CH2)3
and (CH2)6 (8b-c) against MCF-7 and HL-60 cells. For 12a–c, the linkage of 4
carbons (12c) was more beneficial to antiproliferative activity than 2 carbons (12a)
and 3 carbons (12b) against HL-60 cells. Comparing the antiproliferative activities of
H2S releasing evodiamine derivatives (8a, 10b, 11b-c and 12a-c) with those of H2S
donors (5 and 7) and evodiamine, it could be found that the hybrids showed better
antiproliferative properties. Furthermore, in order to investigate whether the hybrids
exhibited selective antiproliferative activities between normal cells and malignant
cancer cells, all hybrids were screened against PBMCs with evodiamine as control.
All the target compounds exhibited inconsequential antiproliferative activity (IC50 >
100 µM) in comparison with evodiamine (IC50 20.96 µM). Among these hybrids, 12c
exhibited the most potent activities against HL-60 cells with IC50 value of 0.58 µM
and good selectivity between tumor and normal cells. So, 12c was selected for further
mechanism study to search for a potentially safer chemotherapy agent.
2.2.2 Cell cycle analysis
The effects of 12c on HL-60 cell cycle were first examined. HL-60 cells were

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treated with different concentrations of 12c (0, 0.3, 0.6 and 1.2 µM) for 48 h and
stained with propidium iodide (PI), followed by flow cytometry analysis. Non-treated
cells were used as control. As shown in Fig. 2, the cells in G1, S and G2/M phases of
control group accounted for 65.75%, 24.06% and 10.19%, respectively. After
treatment with different concentrations of 12c for 48 h, the cells of G2/M phase
increased from 10.19% to 11.41%, 16.63% and 22.02%, respectively. The results
indicated that 12c suppressed HL-60 cell proliferation through inducing cell cycle
arrest at G2/M phase.
2.2.3 The morphological analysis by Hoechst 33258 staining
The remarkable changes of morphological features, such as cell shrinkage,
chromatin condensation, the rupture of cell membrane and the nuclear fragmentation
are the characteristics of apoptotic cells [67]. Hoechst 33258, which stains the cell
nuclei and emits fluorescence allowing the visualization of nuclear morphological
changes, is a membrane permeable dye. Thus, Hoechst 33258 staining were carried
out under fluorescent microscope on 12c (0, 0.3, 0.6 and 1.2 µM) treated HL-60 cells
to determine whether apoptosis was triggered during inhibition of cell growth. As
shown in Fig. 3, control cells were consistently stained with Hoechst 33258 and
presented round homogeneous nuclei, without morphological changes, while the 12c
treated HL-60 cells exhibited bright chromatin condensation and nuclear
fragmentation, a sign of apoptosis. The stain results certified apoptosis was involved
in 12c treated HL-60 cells.
2.2.4 Cell apoptosis assay
Apoptosis is a natural process of programmed cell death. However, tumor cells
can divide relentlessly without programmed cell death, which mainly due to the lack
of apoptosis. Thus, chemical induced apoptosis in tumor cells is pivotal to cancer

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therapy [68]. Therefore, to further certify 12c induced apoptosis in HL-60 cells, an
annexin V-FITC/PI binding assay was performed. HL-60 cells were treated with 12c
at indicated concentrations (0, 0.3, 0.6 and 1.2 µM), then the percentages of apoptotic
cells were measured by flow cytometry. As shown in Fig. 4, 48 h after the treatment
of 12c gave substantial rise to the apoptotic ratios from 3.42% of vehicle control to
11.27% (0.3 µM), 35.06% (0.8 µM) and 60.68% (1.2 µM) for different concentrations
applied in a concentration-dependent manner. Aforementioned results demonstrated
that 12c exerted its antiproliferative effects possibly by inducing apoptosis in HL-60
cells.
2.2.5 Mitochondria membrane potential analysis
The mitochondrion is an organelle that plays an essential role in the induction and
control of apoptosis. Its dysfunction, including the loss of mitochondrial membrane
potential, is a key event that takes place during drug-induced apoptosis. Both the inner
and the outer mitochondrial membranes were involved in these changes, which
sequentially lead to dissipate inner transmembrane potential, promote mitochondrial
permeability transition and induce the release of pro-apoptotic proteins [69]. H2S is
also known to induce apoptosis by activating the intrinsic mitochondrion-mediated
pathways [70]. In an effort to disclose the participation of mitochondria pathway in
12c induced apoptosis of HL-60 cells, the changes of mitochondrial membrane
potentials were assessed. HL-60 cells were treated with different concentrations of
12c (0.3, 0.6 and 1.2 µM) and vehicle solvent for 48 h incubation, then staining with
the
lipophilic
cationic
fluorescent
probe
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1). JC-1
is commercially available. In normal cells, JC-1 aggregates in the mitochondria and
spontaneously forms complexes which fluoresce red. In apoptosis or necrotic cells,

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JC-1 exists in the monomeric form and stains the cytosol green. After that, flow
cytometry analysis was established to measure the cells with collapsed mitochondrial
membrane. These results were summarized in Fig. 5, the percentage of apoptotic cells
increased from 7.65% of control, to 13.33%, 21.32% and 35.01%, respectively. It was
clear that hybrid 12c could induce apoptosis in HL-60 cells through mitochondria
related pathway.
2.2.6 Effects on apoptosis-related proteins
Abnormal proliferation of cancer cells are mainly due to damage of cell cycle
regulation. So, disruption of cell cycle by therapeutic agents can lead to tumour
growth arrest and ultimately to apoptosis, contributing to cancer therapy. Previously,
some studies have confirmed cells with CDK1 (cyclin B1/cdc2 complex) inhibitor
would tend to be arrested at the G2/M phase while cells with CDK1 activator would
be favoured to enter and then proceed through mitosis [71]. Furthermore, tumor
suppressor gene p53 is critically involved in cell cycle regulation, DNA repair and
programmed cell death. It can also regulate G2/M transition through downregulating
the intracellular level of cyclin B1 and cdc2 [72]. Recently, it has been demonstrated
that endogenous H2S has a relationship with mitochondria-mediated apoptosis [73].
Mitochondria represent the central checkpoint in propagating apoptotic signalling
pathways, upon exposure to apoptotic stimuli, it can activate apoptosis-related
proteins such as Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) to enter
mitochondria, which induce the mitochondria to release cytochrome c and, in turn,
activate the caspase-9 and -3, finally triggering the execution of apoptosis [74]. To
explore the mediative mechanism of aforementioned apoptosis related proteins behind
12c induced apoptosis in HL-60 cells, correspondent western blot analysis was
performed, and subsequent results were summarized in Fig. 6. After 48 h treatment,

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12c significantly upregulated the expression of p53, together with downregulation of
cyclin B1 and cdc2. It could also upregulate proteins expression of proapoptotic Bax,
caspase-3, -9 and cytochrome c, and suppress of antiapoptotic Bcl-2 protein
expression. Thus, it was illuminated that 12c induced cell apoptosis via the
mitochondria-dependent pathway.
3. Conclusion
To summarize, two novel series of evodiamine derivatives capable of releasing
H2S were designed and synthesized for anticancer therapeutics. The antiproliferative
activities of all the target compounds were tested against human cancer Bel-7402,
MCF-7, SGC-7901, Caco-2 and HL-60 cell lines, and human normal PBMCs. The
most effective one, conjugate 12c exhibited cytotoxicity against HL-60 and Caco-2
cell lines with IC50 values of 0.58 and 2.02 µM, respectively. Amazingly,
antiproliferative evaluation showed that efficacy enhancement accompanied with
improved selectivity. Hence, 12c was chosen for further investigation in order to
reveal the cellular mechanisms in HL-60 cell lines. The results demonstrated that 12c
showed strong effects which could induce cell apoptosis and arrest cell cycle at the
G2/M stage. Moreover, incubation with 12c increased the number of cells with
collapsed mitochondrial membrane potentials at low concentrations, so the
mitochondrial pathway would be involved in apoptosis. Subsequently, western blot
analysis confirmed that 12c induced apoptosis via mitochondrial related pathway,
since 12c increased the expression of Bax, cytochrome c, caspase-9, -3 and p53, and
reduced the relative levels of Bcl-2, cyclin B1 and cdc2. In conclusion, 12c, a potent
compound with H2S releasing ability, offers a good strategy for the discovery of
antitumor agents with high efficiency and selectivity.

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4. Experimental
4.1. Chemistry
Chemicals and solvents were purchased from commercial sources. Evodiamine
was obtained from Zelang Biological Techology Co., Ltd. (Nanjing, China) with the
purity > 95%. Further purification by standard methods were employed when
necessary. 1H NMR and 13C NMR spectra were recorded on a Bruker ARX-400 NMR
spectrometer in the indicated solvents (TMS as internal standard): the values of the
chemical shifts were expressed in
δ values (ppm) and the coupling constants (J) in Hz.
Mass spectra were obtained on Agilent 1100 Ion trap mass spectrometer.
High-resolution mass spectra (HR-MS) were carried out on Agilent Q-TOF B.05.01
(B5125.2).
4.1.1. General procedures to synthesize 2a-d, 4a-c and 6a-c
Compound 1 (91 mg, 0.3 mmol) was dissolved in 5 mL anhydrous DMF. Then
NaH (10 mg, 0.36 mmol) was added to a solution of 1 and stirred at 0 for 0.5 h. The
corresponding bromhydrin was added dropwise to reaction solution by stirring at
room temperature for another 3 h, then the mixture was poured into 10 mL of H2O
and extracted with DCM (10 mL × 3). The organic layer was combined, washed with
water and saturated NaCl solution, sequentially, dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude products 2a-d were purified by column
chromatography (PE/EA 5:1~3:1 v/v).
Compound 1 (91 mg, 0.3 mmol) was dissolved in 5 mL anhydrous DMF. NaH (10
mg, 0.36 mmol) was added to a solution of 1 and stirred at 0
corresponding bromate ethyl ester was added dropwise to reaction solution by stirring
at 80 for another 12 h, then the mixture was poured into 10 mL of H2O and
for 0.5 h. The

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extracted with DCM (10 mL × 3). The organic layer was combined, washed with
water and saturated NaCl solution, sequentially, dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude products 3a-c were purified by column
chromatography (PE/EA 20:1~10:1 v/v) to give 3. Next, compounds 3a-c were
hydrolysed in a solution of methanol/potassium hydroxide (MeOH/KOH) by stirring
at room temperature for 2 h. After completion of the reaction as indicated by TLC, the
mixture was concentrated, then H2O was added and the solution was carefully
acidized with 0.6 M hydrochloric acid. The combined extracts were dried over
filtration, and evaporated to yield 4a-c.
Compound 5 (226 mg, 1 mmol), potassium carbonate (220 mg, 3 mmol) and
corresponding dibromo alkyl reagents were dissolved in 5 mL anhydrous acetone and
heated under reflux for 8 h. After that, the reaction mixture was cooled to room
temperature, evaporated under reduced pressure, and the crude product was purified
by flash chromatography eluting with petroleum ether/ethyl acetate (10:1) to afford
6a-c.
4.1.2. General procedures to synthesize 8a-d, 9a-c, 10a-c, 11a-c and 12a-c
A solution of α-thioctic acid (0.1 mmol), EDCI (0.3 mmol) and compound 2a (40
mg, 0.1 mmol) in anhydrous DCM (5 mL) with catalytic amount of DMAP was
stirred mechanically at room temperature until esterification was complete. The
mixture was poured into 10 mL of H2O, and extracted with DCM (10 mL × 3). The
organic layers were combined, washed with saturated NaCl solution, dried over
anhydrous Na2SO4, and concentrated in vacuo. The residues were purified by
chromatography on silica gel (petroleum ether-ethyl acetate 10:1) to afford 8a (35
mg). The preparation and workup of compounds 8b-d were similar to 8a.
Compounds 9a-c were prepared using a procedure similar to that of 8a except

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ADTOH were used instead of α-thioctic acid. Compounds 4a-c were used instead of
compound 2. Chromatography on silica gel with (petroleum ether-ethyl acetate 8:1) as
eluent gave the products.
A mixture of 4a (18 mg, 0.05 mmol), 6a (20 mg, 0.05 mmol), catalytic amount of
TEA and anhydrous DMF (5 mL) was heated at 80 with stirring for 8 h. After that,
the reaction mixture was cooled, diluted with H2O (10 mL) and extracted with ethyl
acetate (10 mL × 3). The organic layers were combined, washed with saturated NaCl
solution, dried over anhydrous Na2SO4 and concentrated in vacuo. The residues were
purified by chromatography on silica gel (petroleum ether-ethyl acetate 6:1) to afford
10a (15 mg). The preparation and workup of 10b-c, 11a-c and 12a-c were similar to
10a.
4.1.2.1 Compound 8a. Pale yellow solid, yield: 55%. 1H NMR (CDCl3, 400 MHz),
δ (ppm): 8.15 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.51 (td,
J = 7.9, 1.2 Hz, 1H, Ar-H), 7.45 (d, J = 7.9 Hz, 1H, Ar-H), 7.31 (d, J = 7.2 Hz, 1H,
Ar-H), 7.28 (m, 1H, Ar-H), 7.22 (m, 1H, Ar-H), 7.18 (d, J = 7.2 Hz, 1H, Ar-H), 6.02
(s, 1H, N-CH-N), 4.92 (m, 1H, N-CH2), 4.74 (m, 1H, 13-N-CH2), 4.42 (m, 2H,
-COOCH2), 3.46 (m, 1H, 13-N-CH2), 3.21 (m, 1H, N-CH2), 3.15 (m, 1H, -CH2), 3.10
(m, 1H, -CH2), 3.04 (m, 1H, -CH2), 2.89 (m, 1H, -CH2), 2.40 (s, 3H, N-CH3), 2.17 (m,
13
2H, -CH2), 1.85 (m, 1H, -S-CH-), 1.23–1.60 (m, 8H, -CH2); C NMR (CDCl3, 100
MHz), δ (ppm): 173.06, 164.57, 150.92, 137.70, 133.07, 129.08, 128.49, 125.93,
124.56, 124.26, 123.42, 123.03, 120.06, 119.18, 113.95, 109.83, 68.15, 62.95, 56.32,
42.54, 40.26, 39.34, 38.56, 36.62, 34.55, 33.94, 28.64, 24.47, 20.43; HRMS (ESI) m/z
calcd for C29H33N3HO3S2 [M+H]+ 536.2036, found 536.2057.
4.1.2.2 Compound 8b. Pale yellow solid, yield: 66%. 1H NMR (CDCl3, 600 MHz),
δ (ppm): 8.14 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.51 (m,

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1H, Ar-H), 7.38 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.25 (m, 1H, Ar-H), 7.21 (m, 1H,
Ar-H), 7.18 (m, 1H, Ar-H), 5.98 (s, 1H, N-CH-N), 4.91 (m, 1H, N-CH2), 4.54 (m, 1H,
13-N-CH2), 4.28 (m, 1H, 13-N-CH2), 4.17 (m, 1H, -COOCH2), 4.03 (m, 1H,
-COOCH2), 3.54 (m, 1H, N-CH2), 3.20 (m, 2H, -CH2), 3.11 (m, 1H, -CH2), 3.03 (m,
1H, -CH2), 2.89 (m, 1H, -CH2), 2.45 (m, 1H, -CH2), 2.40 (s, 3H, N-CH3), 2.18 (m, 2H,
-CH2), 2.12 (m, 2H, -CH2), 1.89 (m, 1H, -S-CH-), 1.34–1.65 (m, 6H, -CH2); 13C NMR
(CDCl3, 150 MHz) δ (ppm): 173.32, 164.62, 150.98, 137.17, 133.02, 129.07, 128.41,
125.93, 124.47, 124.26, 123.23, 122.88, 119.86, 119.24, 113.45, 109.61, 68.02, 61.61,
56.45, 40.70, 40.35, 39.37, 38.60, 36.48, 34.61, 33.92, 29.25, 28.82, 24.58, 20.42;
HRMS (ESI) m/z calcd for C30H35N3HO3S2 [M+H]+ 550.2193, found 550.2176.
4.1.2.3 Compound 8c. Pale yellow solid, yield: 65%. 1H NMR (CDCl3, 600 MHz),
δ (ppm): 8.13 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.49 (m,
1H, Ar-H), 7.43 (m, 1H, Ar-H), 7.28 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 7.20 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.00 (s, 1H, N-CH-N), 4.90 (m, 1H, N-CH2), 4.56 (m, 1H,
13-N-CH2), 4.28 (m, 1H, 13-N-CH2), 4.08 (m, 1H, -COOCH2), 4.03 (m, 1H,
-COOCH2), 3.55 (m, 1H, N-CH2), 3.42 (m, 1H, -CH2-O-CH2), 3.37 (m, 2H,
-CH2-O-CH2), 3.34 (m, 1H, -CH2-O-CH2), 3.18 (m, 2H, -CH2), 3.10 (m, 1H, -CH2),
3.03 (m, 1H, -CH2), 2.89 (m, 1H, -CH2), 2.45 (m, 1H, -CH2), 2.40 (s, 3H, N-CH3),
2.30 (m, 2H, -CH2), 2.06 (m, 2H, -CH2), 1.89 (m, 1H, -S-CH-), 1.62–1.77 (m,
6H,-CH2), 1.47 (m, 2H, -CH2); 13C NMR (CDCl3, 150 MHz) δ (ppm): 173.51, 164.69,
151.02, 137.35, 132.94, 128.98, 128.62, 125.79, 124.18, 124.13, 123.13, 122.68,
119.66, 119.05, 113.16, 109.90, 67.92, 67.65, 67.39, 61.51, 56.45, 40.68, 40.33, 39.41,
38.57, 36.39, 34.69, 34.13, 30.29, 29.06, 28.87, 24.78, 20.48; HRMS (ESI) m/z calcd
for C33H41N3NaO4S2 [M+Na]+ 630.2431, found 630.2501.
4.1.2.4 Compound 8d. Pale yellow solid, yield: 59%. 1H NMR (CDCl3, 400 MHz),

ACCEPTED MANUSCRIPT
δ (ppm): 8.15 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.50 (m,
1H, Ar-H), 7.39 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 7.19 (m, 1H,
Ar-H), 7.16 (m, 1H, Ar-H), 5.97 (s, 1H, N-CH-N), 4.91 (m, 1H, N-CH2), 4.37 (m, 1H,
13-N-CH2), 4.19 (m, 1H, 13-N-CH2), 4.03 (m, 2H, -COOCH2), 3.55 (m, 1H, -CH2),
3.21 (m, 1H, N-CH2), 3.15 (m, 1H, -CH2), 3.10 (m, 1H, -CH2), 3.03 (m, 1H, -CH2),
2.89 (m, 1H, -CH2), 2.45 (m, 1H, -CH2), 2.40 (s, 3H, N-CH3), 2.28 (m, 2H, -CH2),
1.90 (m, 2H, -CH2), 1.84 (s, 1H, -S-CH-), 1.38–1.73 (m, 12H, -CH2); 13C NMR
(CDCl3, 100 MHz) δ (ppm): 173.53, 164.58, 150.95, 137.19, 132.96, 129.01, 128.33,
125.75, 124.24, 124.15, 123.08, 122.62, 119.57, 119.06, 113.10, 109.80, 68.12, 64.22,
56.38, 43.89, 40.26, 39.37, 38.51, 36.51, 34.62, 34.10, 30.13, 28.78, 28.59, 26.88,
25.80, 24.73, 20.42; HRMS (ESI) m/z calcd for C33H41N3HO3S2 [M+H]+ 592.2662,
found 592.2677.
1
4.1.2.5 Compound 9a. Brownish red solid, yield: 70%. H NMR (CDCl3, 400
MHz) δ (ppm): 8.15 (d, J = 7.8 Hz, 1H, Ar-H), 7.66 (m, 3H, Ar-H), 7.52 (m, 2H,
Ar-H), 7.36 (m, 3H, Ar-H), 7.30 (m, 1H, Ar-H), 7.25 (m, 1H, Ar-H), 7.23 (s, 1H,
4H-1,2-dithiole-3-thione), 7.17 (d, J = 7.8 Hz, 1H, Ar-H), 6.00 (s, 1H, N-CH-N), 5.44
(m, 1H, 13-N-CH2), 5.25 (m, 1H, 13-N-CH2), 4.94 (m, 1H, N-CH2), 3.26 (m, 1H,
N-CH2), 3.06 (m, 1H, -CH2), 2.94 (m, 1H, -CH2), 2.44 (s, 3H, N-CH3); 13C NMR
(CDCl3, 100 MHz) δ (ppm): 215.64, 171.23, 166.77, 164.59, 153.13, 151.22, 137.94,
136.32, 133.20, 131.04, 129.83, 129.29, 128.99, 128.44 (×2), 126.29, 125.10, 123.69,
123.61, 122.55 (×2), 120.80, 119.57, 114.74, 109.08, 68.33, 65.71, 45.54, 39.42,
20.35; MS (ESI) m/z calcd for C30H23N3HO3S3 [M+H]+ 569.1, found 569.8.
1
4.1.2.6 Compound 9b. Brownish red solid, yield: 30%. H NMR (CDCl3, 400
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.72 (m, 1H, Ar-H), 7.61 (d, J =
8.5 Hz, 2H, Ar-H), 7.53 (m, 1H, Ar-H), 7.50 (m, 1H, Ar-H), 7.46 (m, 1H, Ar-H), 7.38

ACCEPTED MANUSCRIPT
(s, 1H, 4H-1,2-dithiole-3-thione), 7.31 (m, 1H, Ar-H), 7.21 (m, 2H, Ar-H), 7.06 (d, J
= 8.5 Hz, 2H, Ar-H), 6.01 (s, 1H, N-CH-N), 4.92 (m, 1H, N-CH2), 4.57 (m, 1H,
13-N-CH2), 4.36 (m, 1H, 13-N-CH2), 3.20 (m, 1H, N-CH2), 3.04 (m, 1H, -CH2), 2.91
(m, 1H, -CH2), 2.65 (m, 2H, -CH2), 2.41 (s, 3H, N-CH3), 2.30 (m, 2H, -CH2); 13C
NMR (CDCl3, 100 MHz) δ (ppm): 215.64, 171.61, 170.79, 164.67, 153.38, 150.95,
137.41, 136.18, 133.10, 131.04, 129.41, 129.17, 128.97, 128.32 (×2), 125.96, 124.50,
123.30, 123.11, 122.82 (×2), 120.02, 119.31, 113.85, 109.82, 68.21, 65.70, 50.97,
42.90, 31.60, 25.14, 20.49; HRMS (ESI) m/z calcd for C32H27N3NaO3S3 [M+Na]+
620.1040, found 620.1107.
1
4.1.2.7 Compound 9c. Brownish red solid, yield: 46%. H NMR (CDCl3, 400
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.64 (d, J = 8.7 Hz, 2H, Ar-H),
7.61 (m, 1H, Ar-H), 7.49 (m, 1H, Ar-H), 7.40 (m, 1H, Ar-H), 7.38 (s, 1H,
4H-1,2-dithiole-3-thione), 7.29 (m, 1H, Ar-H), 7.17–7.24 (m, 3H, Ar-H), 7.13 (d, J =
8.7 Hz, 2H, Ar-H), 5.99 (s, 1H, N-CH-N), 4.91 (m, 1H, N-CH2), 4.45 (m, 1H,
13-N-CH2), 4.28 (m, 1H, 13-N-CH2), 3.20 (m, 1H, N-CH2), 3.03 (m, 1H, -CH2), 2.90
(m, 1H, -CH2), 2.62 (m, 2H, -CH2), 2.41 (s, 3H, N-CH3), 1.98 (m, 2H, -CH2), 1.82 (m,
2H, -CH2); 13C NMR (CDCl3, 100 MHz) δ (ppm): 215.60, 171.64, 171.07, 164.65,
153.53, 150.99, 137.25, 136.15, 133.08, 129.35, 129.12, 128.37, 128.32 (×2), 125.92,
124.46, 124.25, 123.25, 122.89 (×3), 119.84, 119.27, 113.49, 109.78, 68.24, 43.63,
39.43, 36.63, 33.94, 29.64, 22.46, 20.48; HRMS (ESI) m/z calcd for C33H29N3NaO3S3
[M+Na]+ 634.1263, found 634.1219.
1
4.1.2.8 Compound 10a. Brownish red solid, yield: 50%. H NMR (CDCl3, 400
MHz) δ (ppm): 8.12 (d, J = 7.8 Hz, 1H, Ar-H), 7.71 (m, 1H, Ar-H), 7.61 (m, 1H,
Ar-H), 7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.52 (m, 1H, Ar-H), 7.44 (m, 1H, Ar-H), 7.39
(s, 1H, 4H-1,2-dithiole-3-thione), 7.24 (m, 1H, Ar-H), 7.19 (m, 1H, Ar-H), 7.13 (d, J

ACCEPTED MANUSCRIPT
= 7.8 Hz, 1H, Ar-H), 6.86 (d, J = 8.8 Hz, 2H, Ar-H), 5.93 (s, 1H, N-CH-N), 5.28 (m,
1H, 13-N-CH2), 5.02 (m, 1H, 13-N-CH2), 4.90 (m, 1H, N-CH2), 4.57 (m, 1H,
O-CH2-CH2-O), 4.49 (m, 1H, O-CH2-CH2-O), 4.18 (m, 2H, O-CH2-CH2-O), 3.17 (m,
1H, N-CH2), 3.00 (m, 1H, -CH2), 2.90 (m, 1H, -CH2), 2.39 (s, 3H, N-CH3); 13C NMR
(CDCl3, 100 MHz) δ (ppm): 215.34, 172.67, 168.69, 164.50, 161.49, 150.92, 137.88,
135.01, 133.02, 131.03, 129.14, 128.96, 128.79 (×2), 126.16, 124.95, 124.81, 123.55,
123.37, 120.50, 119.37, 115.51 (×2), 114.31, 109.15, 68.17, 66.07, 65.68, 63.44,
45.30, 30.70, 20.33; HRMS (ESI) m/z calcd for C32H27N3NaO4S3 [M+Na]+ 636.1056,
found 636.1002.
1
4.1.2.9 Compound 10b. Brownish red solid, yield: 20%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (d, J = 7.8 Hz, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.58 (d, J = 8.6
Hz, 2H, Ar-H), 7.48 (m, 1H, Ar-H), 7.43 (m, 1H, Ar-H), 7.37 (s, 1H,
4H-1,2-dithiole-3-thione), 7.28 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 7.19 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.93 (d, J = 8.6 Hz, 2H, Ar-H), 5.94 (s, 1H, N-CH-N),
4.90 (m, 1H, N-CH2), 4.43 (m, 2H, O-CH2-CH2-O), 4.26 (m, 2H, O-CH2-CH2-O),
4.12 (m, 2H, 13-N-CH2), 3.18 (m, 1H, N-CH2), 3.01 (m, 1H, -CH2), 2.88 (m, 1H,
-CH2), 2.42 (m, 2H, -CH2), 2.38 (s, 3H, N-CH3), 2.18 (m, 2H, -CH2); 13C NMR
(CDCl3, 150 MHz) δ (ppm): 215.29, 172.79, 172.60, 164.63, 161.70, 150.96, 137.34,
134.94, 133.04, 129.10, 128.75 (×2), 128.34, 128.24, 125.86, 124.80, 124.48, 123.31,
122.94, 119.88, 119.20, 115.58 (×2), 113.58, 109.85, 68.12, 66.15, 62.64, 53.57,
43.02, 39.38, 31.46, 25.30, 20.47; HRMS (ESI) m/z calcd for C34H31N3NaO4S3
[M+Na]+ 664.1369, found 664.1323.
1
4.1.2.10 Compound 10c. Brownish red solid, yield: 20%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (d, J = 7.8 Hz, 1H, Ar-H), 7.60 (d, J = 7.8 Hz, 1H, Ar-H), 7.56 (d,
J = 8.6 Hz, 2H, Ar-H), 7.49 (m, 1H, Ar-H), 7.37 (m, 1H, Ar-H), 7.36 (s, 1H,

ACCEPTED MANUSCRIPT
4H-1,2-dithiole-3-thione), 7.28 (m, 1H, Ar-H), 7.24 (m, 1H, Ar-H), 7.21 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.91 (d, J = 8.6 Hz, 2H, Ar-H), 5.95 (s, 1H, N-CH-N),
4.90 (m, 1H, N-CH2), 4.41 (m, 2H, O-CH2-CH2-O), 4.37 (m, 1H, 13-N-CH2), 4.21 (m,
1H, 13-N-CH2), 4.17 (m, 2H, O-CH2-CH2-O), 3.16 (m, 1H, N-CH2), 3.01 (m, 1H,
-CH2), 2.88 (m, 1H, -CH2), 2.40 (m, 2H, -CH2), 2.38 (s, 3H, N-CH3), 1.87 (m, 2H,
13
-CH2), 1.73 (m, 2H, -CH2); C NMR (CDCl3, 150 MHz) δ (ppm): 215.29, 173.07,
172.08, 164.66, 161.74, 151.00, 137.21, 134.93, 133.07, 129.10, 128.73 (×2), 128.37,
128.22, 125.86, 124.77, 124.44, 123.30, 122.80, 119.78, 119.22, 115.57 (×2), 113.34,
109.82, 68.17, 66.26, 62.44, 43.68, 39.41, 36.60, 33.73, 29.68, 22.54, 20.48; HRMS
(ESI) m/z calcd for C35H33N3NaO4S3 [M+Na]+ 678.1525, found 678.1465.
1
4.1.2.11 Compound 11a. Brownish red solid, yield: 38%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.12 (d, J = 7.8 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H), 7.56 (d,
J = 8.8 Hz, 2H, Ar-H), 7.45 (m, 1H, Ar-H), 7.39 (s, 1H, 4H-1,2-dithiole-3-thione),
7.25(m, 1H, Ar-H), 7.24 (m, 1H, Ar-H), 7.22 (m, 1H, Ar-H), 7.19 (m, 1H, Ar-H), 7.14
(m, 1H, Ar-H), 6.79 (d, J = 8.8 Hz, 2H, Ar-H), 5.91 (s, 1H, N-CH-N), 5.21 (m, 1H,
13-N-CH2), 4.97 (m, 1H, 13-N-CH2), 4.89 (m, 1H, N-CH2), 4.35 m, 2H,
O-CH2-CH2-O), 3.83 (m, 2H, O-CH2-CH2-O), 3.17 (m, 1H, N-CH2), 2.97 (m, 1H,
-CH2), 2.87 (m, 1H, -CH2), 2.37 (s, 3H, N-CH3), 2.07 (m, 2H, -CH2); 13C NMR
(CDCl3, 150 MHz) δ (ppm): 215.23, 172.97, 168.65, 164.52, 161.92, 150.87, 137.84,
134.80, 133.07, 129.12, 128.67 (×2), 128.14, 126.08, 124.81, 124.45, 123.52, 123.35,
120.46, 119.36, 115.42 (×2), 115.19, 114.20, 109.15, 68.07, 64.22, 62.04, 45.43,
39.31, 36.81, 28.34, 20.30; HRMS (ESI) m/z calcd for C33H29N3NaO4S3 [M+Na]+
650.1212, found 650.1160.
1
4.1.2.12 Compound 11b. Brownish red solid, yield: 26%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H),

ACCEPTED MANUSCRIPT
7.56 (d, J = 8.8 Hz, 2H, Ar-H), 7.49 (m, 1H, Ar-H), 7.43 (m, 1H, Ar-H), 7.36 (s, 1H,
4H-1,2-dithiole-3-thione), 7.28 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 7.19 (m, 1H,
Ar-H), 7.16 (m, 1H, Ar-H), 6.92 (d, J = 8.8 Hz, 2H, Ar-H), 5.95 (s, 1H, N-CH-N),
4.90 (m, 1H, N-CH2), 4.43 (m, 1H, O-CH2-CH2-O), 4.24 (m, 2H, O-CH2-CH2-O),
4.14 (m, 1H, O-CH2-CH2-O), 4.02 (m, 2H, 13-N-CH2), 3.17 (m, 1H, N-CH2), 3.02(m,
1H, -CH2), 2.88 (m, 1H, -CH2), 2.38 (s, 3H, N-CH3), 2.35 (m, 2H, -CH2), 2.16 (m, 2H,
13
-CH2), 2.04 (m, 2H, -CH2); C NMR (CDCl3, 150 MHz) δ (ppm): 215.16, 172.99,
172.68, 164.62, 162.09, 150.94, 137.30, 134.73, 133.02, 129.05, 128.68 (×2), 128.29,
128.15, 125.82, 124.41, 124.19, 123.26, 122.90, 119.84, 119.17, 115.46 (×2), 113.52,
109.84, 68.08, 64.79, 61.32, 43.00, 39.35, 36.53, 31.43, 28.42, 25.27, 20.44; HRMS
(ESI) m/z calcd for C35H33N3NaO4S3 [M+Na]+ 678.1525, found 678.1458.
1
4.1.2.13 Compound 11c. Brownish red solid, yield: 14%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.60 (m, 1H, Ar-H), 7.56 (d, J =
8.8 Hz, 2H, Ar-H), 7.49 (m, 1H, Ar-H), 7.39 (m, 1H, Ar-H), 7.37 (s, 1H,
4H-1,2-dithiole-3-thione), 7.28 (m, 1H, Ar-H), 7.23 (m, 1H, Ar-H), 7.21 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.91 (d, J = 8.8 Hz, 2H, Ar-H), 5.95 (s, 1H, N-CH-N),
4.90 (m, 1H, N-CH2), 4.38 (m, 1H, O-CH2-CH2-O), 4.23 (m, 2H, O-CH2-CH2-O),
4.18 (m, 1H, O-CH2-CH2-O), 4.04 (m, 2H, 13-N-CH2), 3.17 (m, 1H, N-CH2), 3.02(m,
1H, -CH2), 2.88 (m, 1H, -CH2), 2.37 (s, 3H, N-CH3), 2.34 (m, 2H, -CH2), 2.08 (m, 2H,
-CH2), 1.86 (m, 2H, -CH2), 1.70 (m, 2H, -CH2); 13C NMR (CDCl3, 150 MHz) δ (ppm):
215.24, 173.13, 173.01, 164.66, 162.13, 151.00, 137.20, 134.80, 133.07, 131.05,
129.10, 128.70 (×2), 128.37, 128.17, 125.86, 124.43, 123.28, 122.79, 119.77, 119.22,
115.49 (×2), 113.32, 109.80, 68.16, 64.81, 61.11, 43.69, 39.41, 36.59, 33.82, 30.68,
28.53, 22.58, 20.47; HRMS (ESI) m/z calcd for C36H35N3HO4S3 [M+H]+ 670.1862,
found 670.1860.

ACCEPTED MANUSCRIPT
1
4.1.2.14 Compound 12a. Brownish red solid, yield: 34%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.13 (dd, J = 7.8, 1.2 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H),
7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.48 (m, 1H, Ar-H), 7.38 (s, 1H,
4H-1,2-dithiole-3-thione), 7.29 (m, 2H, Ar-H), 7.24 (m, 1H, Ar-H), 7.20 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.89 (d, J = 8.8 Hz, 2H, Ar-H), 5.94 (s, 1H, N-CH-N),
5.20 (m, 1H, 13-N-CH2), 4.97 (m, 1H, 13-N-CH2), 4.90 (m, 1H, N-CH2), 4.22 (m, 2H,
O-CH2-CH2-O), 3.91 (m, 2H, O-CH2-CH2-O), 3.23 (m, 1H, N-CH2), 3.02 (m, 1H,
-CH2), 2.89 (m, 1H, -CH2), 2.41 (s, 3H, N-CH3), 1.71–1.81 (m, 4H, -CH2); 13C NMR
(CDCl3, 150 MHz) δ (ppm): 215.18, 173.08, 168.79, 164.56, 162.23, 150.91, 137.87,
134.72, 133.03, 129.10, 128.69 (×2), 128.16, 126.09, 124.74, 124.28, 123.51, 123.30,
120.41, 119.33, 115.45 (×2), 115.21, 114.17, 109.17, 68.09, 67.56, 65.10, 45.37,
39.34, 36.83, 29.80, 25.40, 20.31; HRMS (ESI) m/z calcd for C34H31N3NaO4S3
[M+Na]+ 664.1369, found 664.1388.
1
4.1.2.15 Compound 12b. Brownish red solid, yield: 10%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.61 (d, J = 7.8 Hz, 1H, Ar-H),
7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.49 (m, 1H, Ar-H), 7.43 (m, 1H, Ar-H), 7.38 (s, 1H,
4H-1,2-dithiole-3-thione), 7.29 (m, 1H, Ar-H), 7.24 (m, 1H, Ar-H), 7.20 (m, 1H,
Ar-H), 7.17 (m, 1H, Ar-H), 6.93 (d, J = 8.8 Hz, 2H, Ar-H), 5.96 (s, 1H, N-CH-N),
4.90 (m, 1H, N-CH2), 4.45 (m, 1H, O-CH2-CH2-O), 4.26 (m, 1H, O-CH2-CH2-O),
4.10 (m, 1H, O-CH2-CH2-O), 4.02 (m, 1H, O-CH2-CH2-O), 3.99 (m, 2H, 13-N-CH2),
3.19 (m, 1H, N-CH2), 3.03 (m, 1H, -CH2), 2.89 (m, 1H, -CH2), 2.39 (s, 3H, N-CH3),
2.36 (m, 2H, -CH2), 2.17 (m, 2H, -CH2), 1.81 (m, 2H, -CH2), 1.75 (m, 2H, -CH2); 13C
NMR (CDCl3, 150 MHz) δ (ppm): 215.24, 173.12, 172.80, 164.66, 162.33, 150.98,
137.35, 134.75, 133.04, 129.10, 128.72 (×2), 128.34, 128.18, 125.86, 124.43, 124.29,
123.29, 122.93, 119.86, 119.19, 115.50 (×2), 113.55, 109.88, 68.12, 67.76, 64.27,

ACCEPTED MANUSCRIPT
43.07, 39.39, 36.57, 31.51, 25.77, 25.40, 25.34, 20.48. HRMS (ESI) m/z calcd for
C36H35N3NaO4S3 [M+Na]+ 692.1682, found 692.1617.
1
4.1.2.16 Compound 12c. Brownish red solid, yield: 5%. H NMR (CDCl3, 600
MHz) δ (ppm): 8.14 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 7.60 (d, J = 7.8 Hz, 1H, Ar-H),
7.59 (d, J = 8.8 Hz, 2H, Ar-H), 7.50 (m, 1H, Ar-H), 7.38 (s, 1H,
4H-1,2-dithiole-3-thione), 7.37 (m, 1H, Ar-H), 7.28 (m, 1H, Ar-H), 7.24 (m, 1H,
Ar-H), 7.22 (m, 1H, Ar-H), 7.17 (m, 1H, Ar-H), 6.93 (d, J = 8.8 Hz, 2H, Ar-H), 5.96
(s, 1H, N-CH-N), 4.90 (m, 1H, N-CH2), 4.39 (m, 1H, O-CH2-CH2-O), 4.20 (m, 1H,
O-CH2-CH2-O), 4.11 (m, 2H, O-CH2-CH2-O), 4.01 (m, 2H, 13-N-CH2), 3.18 (m, 1H,
N-CH2), 3.02(m, 1H, -CH2), 2.88 (m, 1H, -CH2), 2.39 (s, 3H, N-CH3), 2.33 (m, 2H,
-CH2), 1.76-1.90 (m, 8H, -CH2); 13C NMR (CDCl3, 150 MHz) δ (ppm): 215.26,
173.24, 173.13, 164.69, 162.36, 151.02, 137.22, 134.77, 133.08, 129.11, 128.73 (×2),
128.39, 128.19, 125.87, 124.44, 124.30, 123.30, 122.79, 119.76, 119.22, 115.51 (×2),
113.33, 109.83, 68.18, 67.78, 64.06, 43.70, 39.42, 36.61, 33.91, 29.77, 25.78, 25.47,
22.65, 20.49; HRMS (ESI) m/z calcd for C37H37N3HO4S3 [M+H]+ 684.2019, found
684.1935.
4.2 MTT assay
Antiproliferative activity was examined by the MTT assay following the method
described previously [75,76]. The assay was performed in 96-well plates. Cells were
added to each well and incubated for 24 h at 37 oC in a humidified atmosphere of 5%
CO2. Then cells were incubated in the presence or absence of test compounds. After
72 h, MTT solution (20 µL/well, 5 mg/mL) was added to each cultured medium,
which was incubated for another 4 h. DMSO (150 µL/well) was added to each well
sequentially, 10 minutes of incubation at room temperature was followed. After 10
min, the OD of each well was measured on a Microplate Reader (BIO-RAD) at the

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wavelength of 570 nm. In these experiments, the negative reference was 0.1% DMSO
and 5-FU was used as the positive reference. The IC50 values were calculated
according to the inhibition ratios.
4.3 Cell cycle analysis
The cell cycle of HL-60 cell line treated with 12c was evaluated by flow
cytometry with PI (KGA511, KeyGEN Biotech, Nanjing, China). HL-60 cells were
o
seeded in 6-well plates and incubated at 37 C for 24 h. Then, 12c was dissolved in
DMSO and added indicated concentrations for incubation in triplicate manner. Blank
solvent of DMSO was added to control cells. After 48 h incubation, all cells were
o
centrifuged and fixed in 70% ethanol at 4 C overnight and suspended again in PBS
mixed with 100 µL RNase A and 400 µL PI. Cell cycle distribution of DNA content
was assessed on a flow cytometer (FACS Calibur Becton-Dickinson, Franklin Lake,
NJ, USA) [77].
4.4 Hoechst 333258 staining
The nuclear morphological modifications were analyzed by fluorescent
microscopy using Hoechst staining. In this experiment, HL-60 cells were seeded on
6-well plates, with 2 mL of medium. After 24 h of incubation, 12c was added at
indicated concentrations and cells were incubated again for a period of 48 h. Cells
were harvested by mild trypsinization, collected by centrifugation and washed twice
with PBS. Cells were then stained with 500 µL of Hoechst solution (2 mg/mL in PBS)
(Keygen, Nanjing, China) for 15 min at room temperature, in darkness. After
incubation, cells were washed with PBS, mounted in a slide and observed with a
fluorescent microscope with a DAPI filter [78].
4.5 Cell apoptosis assay
Apoptosis was analyzed using Annexin V and PI double staining by flow

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cytometry according to the manufacturer's instructions (KGA1024, KeyGEN Biotech,
Nanjing, China) in order to detect apoptotic cells. The HL-60 cells were seeded in
6-well plates overnight, then different concentrations of 12c dissolved in DMSO were
added in triplicate for 24 h incubation. After that, PBS was used twice to wash
incubated cells, then resuspended in annexin V binding buffer. Annexin V-FITC was
o
added to the mixture before 15 minutes incubation in total dark at 25 C. PI was
applied right before acquisition. The percentage of cells positive for PI and/or
Annexin V-FITC was reported inside the quadrants [79,80].
4.6 Mitochondrial membrane potential assay
HL-60 cells were incubated with target molecule 12c (0.3, 0.6 and 1.2 µM) or
vehicle solvent briefly in triplicate fashion for 48 h, then washed with PBS and
stained with JC-1 dye under dark conditions according to the manufacturer’s
instructions (KeyGen Biotech, KGA601). The percentage shares of cells with healthy
or collapsed mitochondrial membrane potentials were monitored through flow
cytometry analysis [81].
4.7 Western blot assay
HL-60 cells were incubated with indicated doses (0.3, 0.6 and 1.2 µM) of 12c in
triplicate for 72 h. The cells were harvested and lysed using lysis buffer, and the
o
solution was centrifuged for 10 min at 4 C. After determination of protein
concentrations, individual cell lysates were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (10% gel, SDS-PAGE) and transferred onto
nitrocellulose membranes. Target proteins in the membranes were blocked with 5%
fat-free milk, and probed with monoclonal antibodies against Bcl-2, Bax, cytochrome
c, caspase-9, -3, p53, cyclin B1, cdc2 and GAPDH (KeyGEN Biotech, Nanjing, China)
o
at 4 C overnight, respectively. The bound antibodies were detected by appropriate

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second antibodies and visualized using an enhanced chemiluminescent reagent. The
relative optical densities of each protein to control GAPDH were determined by
densimetric scanning [82].
Acknowledgment
This paper was financially supported by the Project Funded by China Post
Doctoral Science Foundation (2017T100186), Natural Science Foundation of
Liaoning Province (20170540858), General Scientific Research Projects of
Department of Education in Liaoning Province (2017LQN05), and Career
Development Support Plan for Young and Middle-aged Teachers in Shenyang
Pharmaceutical University.
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