Synthesis of Dithiolethiones and Identification of Potential Neuroprotective Agents via Activation of Nrf2-Driven Antioxidant Enzymes

Article abstract of DOI:10.1021/acs.jafc.9b06360

Oxidative stress is implicated in the pathogenesis of a wide variety of neurodegenerative disorders, and accordingly, dietary supplement of exogenous antioxidants or/and upregulation of the endogenous antioxidant defense system are promising for therapeutic intervention or chemoprevention of neurodegenerative diseases. Nrf2, a master regulator of the cellular antioxidant machinery, cardinally participates in the transcription of cytoprotective genes against oxidative/electrophilic stresses. Herein, we report the synthesis of 59 structurally diverse dithiolethiones and evaluation of their neuroprotection against 6-hydroxydopamine-or H₂O₂-induced oxidative damages in PC12 cells, a neuron-like rat pheochromocytoma cell line. Initial screening identified compounds 10 and 11 having low cytotoxicity but conferring remarkable protection on PC12 cells from oxidative-mediated damages. Further studies demonstrated that both compounds upregulated a battery of antioxidant genes as well as corresponding genes' products. Significantly, silence of Nrf2 expression abolishes cytoprotection of 10 and 11, indicating targeting Nrf2 activation is pivotal for their cellular functions. Taken together, the two lead compounds discovered here with potent neuroprotective functions against oxidative stress via Nrf2 activation merit further development as therapeutic or chemopreventive candidates for neurodegenerative disorders.

Full text of DOI:10.1021/acs.jafc.9b06360

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Functional Structure/Activity Relationships
Synthesis of Dithiolethiones and Identification of Potential
Neuroprotective Agents via Activation of Nrf2-Driven Antioxidant Enzymes
Zi-Long Song, Feifei Bai, Baoxin Zhang, and Jianguo Fang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06360 • Publication Date (Web): 27 Jan 2020
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Synthesis of Dithiolethiones and Identification of Potential Neuroprotective
Agents via Activation of Nrf2-Driven Antioxidant Enzymes
Zi-Long Song, Feifei Bai, Baoxin Zhang, Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry & College of Chemistry and Chemical
Engineering, Lanzhou University.
*Corresponding author, Email: fangjg@lzu.edu.cn.
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Abstract
Oxidative stress is implicated in the pathogenesis of a wide variety of neurodegenerative disorders, and accordingly,
dietary supplement of exogenous antioxidants or/and upregulation of endogenous antioxidant defense system is
promising for therapeutic intervention or chemoprevention of neurodegenerative diseases. Nrf2, a master regulator
of the cellular antioxidant machinery, cardinally participates in the transcription of cytoprotective genes against
oxidative/electrophilic stresses. Herein, we report the synthesis of 59 structurally diverse dithiolethiones and
evaluation of their neuroprotection against 6-hydroxydopamine- or H O -induced oxidative damages in PC12 cells,
2
2
a neuron-like rat pheochromocytoma cell line. Initial screening identified compounds 10 and 11 having low
cytotoxicity but conferring remarkable protection on PC12 cells from oxidative-mediated damages. Further studies
demonstrated that both compounds upregulated a battery of antioxidant genes as well as corresponding genes’
products. Significantly, silence of Nrf2 expression abolishes cytoprotection of 10 and 11, indicating targeting Nrf2
activation is pivotal for their cellular functions. Taken together, the two lead compounds discovered here with
potent neuroprotective functions against oxidative stress via Nrf2 activation merit further development as
therapeutic or chemopreventive candidates for neurodegenerative disorders.
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1. Introduction
Reactive oxygen species (ROS) are a wide variety of reactive molecules that are produced, transformed, and
consumed as a consequence of aerobic metabolism in living organisms. Under physiological conditions, ROS are
maintained at a low level and play critical roles in cellular signaling; however, when the ROS homeostasis is
disturbed with a favor of ROS accumulation, generally arising from excessive ROS generation or/and impairment
of antioxidant defense, oxidative stress occurs.1-3 Oxidative stress causes deleterious effects on biomolecules, which
ultimately contributes to many pathophysiological conditions,4 such as cancer, cardiovascular diseases and
neurodegenerative disorders.
The central nervous system (CNS) is particularly susceptible to oxidative stress due to its high oxygen consumption,
remarkable levels of transition metals, and enrichment of polyunsaturated fatty acids, rendering it more vulnerable
to ROS-mediated damage.4-7 Furthermore, dopaminergic neurons may suffer additional oxidative burden owing to
auto-oxidation and metabolism of dopamine, resulting in electrophilic dopamine quinones (DAQs). The subsequent
metabolites of DAQs as well as DAQs themselves are able to consume reducing equivalents NADPH, react with
vital nucleic acid, and deplete thiol antioxidant glutathione (GSH), all of which processes are accompanied by
8
concomitant production of large amounts of ROS. As a consequence, neurodegenerative diseases, such as
Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis
(ALS), are all considered to be associated with oxidative stress.5-10 Given the fact that elevated oxidative stress and
the progressive loss of neuronal cell populations are characteristics of the development of the neurodegenerative
diseases, alleviating oxidative stress is a reasonable strategy to ameliorate the symptoms or arrest/reverse the
progression of these diseases.6, 8 As a result, potential therapeutic pharmacological interventions based on charging
exogenous or/and endogenous antioxidants have been developed during the past years. Compared to supplement of
exogenous antioxidants, activation of endogenous antioxidant defense pathway has emerged as a preferred strategy
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in mitigating the detrimental effects of ROS as such regulation is usually at the transcriptional level and
sustainable.11-14
Nuclear factor erythroid 2-related factor 2 (Nrf2), a member of cap ‘n’ collar family of basic region leucine zipper
transcription factors, is a master regulator of antioxidant response signaling. Many antioxidant and detoxifying
enzymes, such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutathione (GSH)
biosynthetic enzymes glutamate cysteine ligase (GCL), thioredoxin 1 (Trx1) and thioredoxin reductase 1 (TrxR1),
are regulated by Nrf2 at the transcription level.15-16 Under basal conditions, Nrf2 is anchored in the cytoplasm to its
repressor protein Keap1 (Kelch-like ECH-associated protein 1), a substrate specific adaptor component of
cullin3-based E3 ubiquitin ligase complexes targeting Nrf2 for ubiquitination and subsequent proteasomal
degradation.17-18 As such, this maintains Nrf2 at a low concentration in nucleus and keeps subsequent transcription
of target genes at a basal level. Upon exposure to stressed conditions, Keap1 acts as a stress sensor on account of its
several hypersensitive cysteine residues that can be covalently modified by ROS and electrophiles,19 giving rise to
dissociation of Nrf2 from Keap1-cullin3-Nrf2 complexes and abrogation of Keap1-dependent depression of Nrf2.
As a result thereafter, Nrf2 translocates to the nucleus and constitutes a heterodimer with other transcription
activators, e.g., small musculoaponeurotic fibrosarcoma (sMAF) proteins, and binds to the enhancer sequence of
antioxidant response elements (AREs) in the regulatory promoter regions of a wide range of cytoprotective genes,
which ultimately initiates the expression of abundant protective genes. On this ground, activation of
Keap1-Nrf2-ARE pathway is promising in chemoprevention of oxidative stress-related diseases or is of therapeutic
potential in treatment of such disorders.11-14, 20-23
2
S
S
S
S
S
S
S
S
S
S
S
S
S
3
1
S
S
N
5
4
N
D3T
CPDT
CHDT
O
Oltipraz
ADT
8
3
4
8
Figure 1. Structures of typical dithiolethiones.
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Small molecules have been continually attracting extensive attention in the discovery and development of
antioxidant agents targeting Keap1-Nrf2-ARE pathway.24-30 Encouragingly, past decades have witnessed
tremendous progress in successful identification of Nrf2 activators, and works by our group also unveiled several
naturally occurring and synthetic molecules that target Keap1-Nrf2-ARE pathway and sequentially protected cells
from oxidative insults.28, 31-37 Recently, numerous lines of evidences demonstrated that 3H-1,2-dithiole-3-thione
(D3T) and its several synthetic derivatives (e.g., ADT, CPDT, CHDT, and Oltipraz) (Figure 1) are of medicinal
application potential. All of these molecules are analogues of naturally occurring dithiolethiones isolated from
cruciferous vegetables and abundant especially in broccoli. These molecules possess protective effects against
carcinogenesis38-43 and play neuroprotective roles in various cell lines and animal models.44-48 Although several of
this class of sulfur-containing heterocyclic molecules represent promising therapeutic or preventive potential in
combating with neurodegenerative disorders, the orchestrating mechanism of action underlying their
neuroprotective effects and their structure-activity relationships (SAR) have not been thoroughly elucidated.47-48
With the intention of advancing our development of novel small molecules with redox-regulating properties, we
reported herein the synthesis of 59 structurally diverse dithiolethiones and evaluation of them as potential
neuroprotective agents against 6-hydroxydopamine (6-OHDA)- or H O -induced oxidative damages in a
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101
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neuron-like rat pheochromocytoma cell line (PC12 cells). After examination of their cytotoxicity and
neuroprotective effects, preliminary SAR was delineated and summarized, which will facilitate further optimization
of dithiolethiones as potential chemotherapeutic or chemopreventive candidates. Two compounds (10 and 11) were
demonstrated to exhibit low cytotoxicity and high cytoprotective activity, and thereby were selected for further
studies. Treatment of PC12 cells with compounds 10 and 11 promoted the upregulation of a variety of Nrf2-driven
antioxidant species (e.g., GSH, HO-1, NQO1, TrxR and Trx). Silence of Nrf2 expression by RNA interference
drastically abolished the cytoprotective effects of compounds 10 and 11, supporting the essential participation of
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Nrf2 for the cellular performance of the agents.
S
O
O
O
O
S
P4S10, S8
O
P4S10, S8, HMDO
S
S
S
pyridine, 130 o
C
xylene, reflux, 1-5 h
n
S
O
O
O
O
n
n=1, b1
n=2, b2
n=1, comp 3
n=2, comp 4
a1
c1
comp 1
S
O
O
O
1
. NaOMe, MeOH
P4S10, S8, HMDO
MeOOC
+
H
O
2. CH COOH,H O
O
O
S
O
O
3
2
xylene, reflux, 0.5 h
S
c2
c3 OH
comp 2
S
S
O
O
S
S
4 10 8
P S , S , HMDO
Br
K2S
DMF, 140 o
R1
O
xylene, reflux, 1-5 h R1
S
C
R1=methyl, e1
R1=phenyl, e2
R1=methyl, comp 5
R1=phenyl, comp 7
S
CHO
d1
comp 6
S
O
O
n
O
O
P4S10, S8, HMDO
xylene, reflux, 1-5 h
NaH, diethyl carbonate
THF, reflux
S
S
n
n
n=1, f1
n=2, f2
n=1, f3
n=2, f4
n=1, comp8
n=2, comp9
S
O
O
S
1
. CDI, THF, reflux
P S , S , HMDO
S
4
10
8
COOH
R2
2. Ethyl potassium malonate, reflux
xylene, reflux, 1-5 h
R2
O
R2
g1
g2
comp 10~43
R2
R2
R2
R2
O
O
O2N
ortho comp 10
meta comp 13
para comp 16
comp 25
comp 32
comp 37
O
F
O
ortho comp 11
meta comp 14
para comp 17
comp 26
comp 33
comp 34
ortho comp 38
meta g3
para g4
O
Cl
Br
ortho comp 27
meta comp 28
para comp 29
NO2
ortho comp 12
meta comp 15
para comp 18
2 2 4
Na S O
O
acetone/H O
2
OCH3
comp 35
ortho comp 41
O
O
O
meta comp 42
para comp 43
NC
ortho comp 19
meta comp 20
para comp 21
comp 30
comp 31
NH2
comp 36
comp 59
CF3
CH3
N
MeOOC
I
comp 39
comp 40
ortho comp 22
meta comp 23
para comp 24
N
O
S
S
S
O
O
O
COOH
COOC2H5
R3
O
O
R3
h3
h5
R3
O
comp 51~57
h1
con. H2SO4
NaH
h6
4 10 8
P S , S , HMDO
COOH C2H5OH, reflux
O
O
COOC2H5
O
S
S
O
O
xylene, reflux, 1-5 h
S
anhydrous THF, reflux
O
O
O
R3
comp 44~50
h4
R3
O
O h2
O
h7
O
O
O
R3
comp 44
comp 51
comp 45
comp 52
comp 46
comp 53
comp 47
comp 54
comp 48
comp 55
comp 49
comp 56
comp 50
comp 57
con. H2SO4
COOH
COOC2H5
O
O
S
C2H5OH, reflux
S
S
LHMDS, -78 o
anhydrous THF
C
OC2H5 P4S10, S8, HMDO
i1
i2
COCl
xylene, reflux, 1-5 h
COOH
SOCl2
reflux
i3
i4
i5
comp 58
1
1
08
09
Scheme 1. General synthetic procedures for various intermediates and target compounds.
1
10
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11 2. Results and Discussion
12 2.1. Chemistry
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16
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18
19
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21
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24
25
26
As is shown in scheme 1, target compounds 1 and 3~7 were synthesized from commercially available chemicals as
starting materials, and compound 2 was synthesized through intermediate c3, which was obtained via the reaction
of c1 with c2 by the method described by Coates and Hobbs.49 Intermediates f3 and f4 were obtained by the
methods described by Che et al.50 and Unsworth et al.51 from commercially available f1 and f2, respectively.
Various commercially available starting materials g1 were converted into intermediates g2 in high yields without
further purification after a concise operation by the method described by Jae et al.52 Key intermediates h6 and h7
5
3
were easily obtained by reactions of various h5 with h3 and h4 . Both h3 and h4 were achieved by ethyl
esterification of h1 and h2 in ethyl alcohol with catalytic amount of concentrated H SO in high yields. Easily
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4
synthetic intermediates i2 and i4 reacted in a condition described by Taylor et al.,54 providing the key intermediate
i5.
Three methods were utilized in the synthesis of 59 target dithiolethiones in this paper, namely, compound 1 by
method of Behringer et al.,55 compound 6 by method of Jin et al.,56 and the other compounds by method of
Curphey.57 All target compounds were characterized through H NMR, 13C NMR and MS. HPLC was employed to
determine the purity of all compounds used for bio-evaluation.
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Figure 2. Cytotoxicity screening of dithiolethiones against (A) PC12 cells and (B) BEAS-2B cells. The cells were seeded in 96-well plates for 24 h,
and then treated with indicated concentrations of compounds for another 24 h. The cell viability was measured by the MTT assay. Representative
graphs are from three independent experiments, with errors bars representing SD values.
1
1
29
30
1
1
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31 2.2. Initial Screening and SAR Studies
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36
37
38
39
40
41
42
As displayed in Figure 2, all target compounds were firstly screened for cytotoxicity against PC12 cells by using
the MTT assay, and compounds 16, 21, 36 and 59 were found to exhibit apparent toxicity toward PC12 cells. Next,
except for compounds 16, 21, 36 and 59, the other 55 target compounds were evaluated for toxicity against
BEAS-2B cells (human bronchial epithelial cell line) by using the same method as used for PC12 cells. Obviously,
the overwhelming majority of the target compounds show little toxicity toward the two cell lines in different
concentrations. Compounds 16, 21, 36 and 59 exhibit apparent toxicity against PC12 cells, while compounds 44~47
possess slight inhibition activity to the growth of BEAS-2B cells. In addition to compounds with distinct
cytotoxicity (comp 16, 21 36, 44~47, and 59), the other compounds were chosen for evaluating their
neuroprotective activity in two well established cellular models of neurodegenerative diseases28, 31-37, 58, and the
levodopa (L-DOPA) is used as the positive compound. Impressively, all compounds are capable of rescuing PC12
cells from 6-OHDA- and H O -induced injury and a large number of compounds are better than L-DOPA in
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preventive effects, with compounds 10 and 11 being the top two most efficacious agents (Figure 3).
1
44
45
1
2 2
Figure 3. Preventive activity screening of dithiolethiones against (A) 6-OHDA- or (B) H O -induced PC12 cell damage. The cells were cultured for 24
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h and subsequently subjected to 20 M indicated compounds or the positive compound L-DOPA for another 24 h. Then the old medium was discarded
2 2
followed by addition of new medium containing (A) 6-OHDA (200 M) or (B) H O (500 M) for another 12 h. The cell viability was evaluated by the
MTT assay. Each bar represents the means ±SD of three independent experiments. **P < 0.01 vs the control group; ^P < 0.05, and ^^P < 0.01 vs the
2 2
6-OHDA or H O treatment.
The preliminary structure-activity relationship (SAR) analysis showed that the structurally simplest compound 1
(D3T) without any substitution have moderate preventive activity. Compound 2 with electron withdrawing group
th
on the 4 position of D3T showed slightly increase in its activity. Five membered ring (compound 3, CPDT) is
th
more favored than six membered ring (compound 4, CHDT). Phenyl substitution (compound 7) at the 5 position
of D3T showed good preventive activity, while compounds 16, 21, 35, 36 and 59 with electron withdrawing groups
on the para position of phenyl exhibit apparent cytotoxicity to PC12 cells, and both 44~50 bearing
3,4,5-trimethoxyphenyl and 58 bearing two phenyl substitutions are somewhat toxic59 to BEAS-2B cells. With
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regard to compounds 10~18, appropriate atom size and electron withdrawing ability play important roles in
preventive activity of these compounds, as chlorine substituted compounds (11, 14 and 17) are the most active
ones. In addition, ortho-halogen substituted compounds showed better activity than meta-halogen substituted ones,
and the para-halogen substituted ones are the least. As for compounds 27~34, the amounts of electron donating
groups make little difference to preventive activity, and the ortho-methoxy group (compound 27) is more favored.
What is more, it is also worth mentioning that n-pentyloxy (compound 34) causes significantly beneficial effect on
its preventive activity. When exploring the effect of chain length on activity of compounds (48~57), we noticed that
th
chain length makes little effect on the activity. Taken together, aromatic substitutions at the 5 position of D3T are
tolerated and favored, and especially the ortho-substituted halogen with suitable electron withdrawing ability plays
th
pivotal roles in improving the preventive activity of compounds. Further studies on optimization of the 4 position
of D3T are underway in our lab.
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1
1
1
1
1
68 2.3. Rescue of PC12 Cells from Oxidative Damages by Compounds 10 and 11
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Because of the better preventive activity and lower cytotoxicity of compounds 10 and 11, they were picked up for
the follow-up studies. 6-OHDA, a neurotoxin, whose toxicity is mainly ascribed to its ability to produce excessive
6
0
amounts of ROS, was applied to generate a cellular model of PD . As depicted in Figure 4A, PC12 cells
challenged with 6-OHDA solely were about half alive in comparison with control cells. When cells were pretreated
with 10 or 11, the 6-OHDA-induced cell death was alleviated remarkably in a dose-dependent manner. Prominently,
compounds 11 rescued PC12 cells up to more than 80% at the concentration of 20 M. H O , a well-known
1
1
1
1
1
2
2
intrinsic signal molecule as well as a kind of ROS species, was used to produce cell neurotoxicity mimicking the
endogenous process of pathogenesis of PD. As shown in Figure 4B, H O treatment leads to roughly 40% death of
2
2
PC12 cells in comparison with the control group. Likewise, pretreatment of PC12 cells with 10 or 11 also markedly
relieved cells from the H O -mediated damage with elevation of cells viability up to impressive 90% at the
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concentration of 20 M. With the intention of confirming the results displayed in Figure 4A and Figure 4B, the
lactate dehydrogenase (LDH) leakage assay was further conducted.28, 31 As displayed in Figure 4C and Figure 4D,
the contents of released LDH in two models were 2-fold more than the control, while this process could be
diminished evidently by pretreatment of the cells with 10 or 11, which was in accordance with the results of MTT
assay. In order to consolidate the results obtained above, we further determined the preventive effect of 10 and 11
in N2a cells (mouse neuroblastoma N2a cells), and the results showed that both 10 and 11 exhibited robust
preventive effect against H O -induced oxidative stress (Figures S1-S3, Supporting Information).
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Given the fact that overproduction of ROS is considered to be persistently implicated in the pathogenesis of a broad
range of both acute and chronic neuropathologies,5, 9-10 we then determined whether compounds 10 and 11 are able
to mitigate ROS generation in PC12 cells caused by 6-OHDA or H O Dichlorodihydrofluorescein diacetate
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2.
(DCFH-DA), a non-fluorescent probe that can be converted into a highly fluorescent molecule by reacting with
ROS in cells, was utilized as a detector probe of ROS. As shown in Figure 5, after treatment of 6-OHDA or H O2
2
solely PC12 cells showed strong fluorescent emission compared with the control groups. However, preincubation
,
of PC12 cells with either 10 or 11 could reduce the fluorescence signal to a great extent, which demonstrates their
ability to alleviate the accumulation of ROS.
1
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1
2 2
Figure 4. Preventive activity of 10 and 11 against 6-OHDA- or H O -induced injury in PC12 cells. (A, B) Protection of PC12 cells from 6-OHDA- or
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2 2
H O -mediated damage by pretreatment with indicated concentrations of 10 and 11. PC12 cells were cultured for 24 h and subsequently exposed to
indicated concentrations of 10 and 11 for another 24 h, and afterwards the old medium was discarded followed by addition of new medium containing
2 2
(A) 6-OHDA (200 M) or (B) H O (500 M) for another 12h. The cell viability was evaluated by the MTT assay. (C, D) Preventive activity of 10 and
11 verified by LDH release assay. PC12 cells were cultured for 24 h, and indicated concentrations of 10 and 11 were added for another 24 h followed
2 2
by addition of (C) 6-OHDA (200 M) or (D) H O (500 M) for further 12 h. Afterwards determination of the activity of released LDH was conducted.
Representative graphs are from three independent experiments, with errors bars representing SD values. **P < 0.01 vs the control group; ^P < 0.05, and
2 2
^^P < 0.01 vs the 6-OHDA or H O treatment.
2
03
04
2
Figure 5. Alleviation of ROS production by 10 and 11 in PC12 cells. The cells were cultured for 24 h, subsequently treated with indicated
2
05
06
2 2
concentrations of 10 and 11 for another 24 h, and then exposed to (A) 6-OHDA or (B) H O for 12 h. ROS levels were determined by DCFH-DA
2
staining.
2
2
2
2
2
07 2.4. Relief of Apoptosis by Compounds 10 and 11
08
09
10
11
Since the cytotoxicity of 6-OHDA and H O to PC12 cells is mainly mediated by induction of apoptotic cell
2 2
death,28, 31 we then asked if compounds 10 and 11 could attenuate 6-OHDA- or H O -induced apoptosis of PC12
2
2
cells. Apoptotic cells are generally characterized by condensed nuclear chromatins, which could be revealed by
fluorescence imaging accomplished with the aid of a DNA staining dye Hoechst 33342. As depicted in Figure 6A
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and Figure 6B, both 6-OHDA and H O lead to apoptosis of PC12 cells, manifested by the occurrence of highly
2 2
fluorescent pycnotic nuclei, while weak fluorescence emission was observed in control groups. Apparently,
pretreatment of PC12 cells with 10 and 11 significantly assuaged cell apoptosis in a dose-dependent manner. To
confirm the results evidenced by cellular morphological changes above, we next determined the activity of
caspase-3, a biochemical indicator of apoptosis. Both 6-OHDA and H O elevated activity of cellular caspase-3 to
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approximately 2.5-fold higher than the control cells, whereas, with addition of compounds 10 or 11, activity of
cellular caspase-3 was ablated conspicuously (Figure 6C and Figure 6D). Finally, we employed flow cytometry to
quantify the cell populations with Annexin V-FITC/PI double staining assay in order to further consolidate the
results obtained above. As shown in scattergrams Figure 7A and 7B and quantification results from Figure 7C and
7D, both 6-OHDA and H O result in death of PC12 cells to a fairly high level. Nevertheless, pretreatment of PC12
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cells with 10 and 11 notably mitigate the oxidative stress-induced insult in a dose-dependent manner. Taken
together, the results above imply that 10 and 11 could relieve PC12 cells of oxidative stress-induced apoptosis.
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Figure 6. Attenuation of 6-OHDA- or H O -induced apoptosis of PC12 cells by 10 and 11. PC12 cells were incubated with indicated concentrations of
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10 and 11 for 24 h, and then were exposed to (A) 6-OHDA or (B) H O for further 12 h. Hoechst 33342 staining assay was employed to visualize and
image apoptotic PC12 cells. Inhibition of activity of caspase-3 by preincubation of PC12 cells with indicated concentrations of 10 and 11 followed by
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exposure to (C) 6-OHDA or (D) H O . The cellular caspase-3 activity was measured by a colorimetric assay. Representative graphs are from three
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independent experiments, with errors bars representing SD values. **P < 0.01 vs the control group. ^^P < 0.01 vs the 6-OHDA or H O treatment.
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Figure 7. Preventive effect of compounds 10 and 11 in PC12 cells analyzed by flow cytometry. PC12 cells were incubated with indicated
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concentrations of 10 or 11 for 24 h followed by (A) 6-OHDA or (B) H O treatment for 12 h, and then the population of live cells, apoptotic cells and
dead cells was analyzed by Annexin V/PI-double-staining assays. The cells are divided into four different populations: Q3 (lower left) is the live cell
population with double-negative staining, Q1 (upper left) in the dead cell population with PI-positive-and Annexin V-negative staining, Q4 (lower right)
is the early apoptotic cell population with Annexin V-positive- and PI-negative staining, Q2 (upper right) is the late apoptotic cell population with
Annexin V/PI-double staining. The quantification results of preventive effect are shown in (C) and (D), and dead cells (Q1), apoptotic cells (Q2 and
Q4), and normal cells (Q4) are illustrated. Representative graphs are from three independent experiments, with errors bars representing SD values. **P
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< 0.01 vs the control group. ^^P < 0.01 vs the 6-OHDA or H O treatment.
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39 2.5. Upregulation of the Antioxidant Defence System by Compounds 10 and 11
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Several studies have demonstrated that certain dithiolethiones exerted their anticarcinogenic or protective effects
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via arousing the antioxidant defence mechanism of Keap1-Nrf2-ARE through covalent modification , oxidation
or persulfidation63 of cysteine sulfhydryls in Keap1. Then we measured whether compounds 10 and 11 could
upregulate the Nrf2-directed antioxidant genes in PC12 cells, such as HO-1, NQO1, TrxR, Trx, GCLC (GCL
catalytic subunit), and GCLM (GCL regulatory subunit). Also, the effects of 10 and 11 on Nrf2 mRNA transcription
level were evaluated. As shown in Figure 8A and Figure 8B, compounds 10 and 11 could boost these detoxifying
genes to a dramatically high level except for Nrf2, with HO-1 being increased to about 10- and 22-fold higher than
the control group, and the mRNA levels reached the peak at 6 h after treatment of PC12 cells with 10 or 11. It is
worth noting that neither 10 nor 11 had any impact on Nrf2 mRNA level, suggesting that Nrf2 gene transcription or
its message stability was not affected by the compounds. Furthermore, the content and activity of the antioxidant
molecules encoded by these genes was determined. As shown in Figure 9, the expression content of the proteins
HO-1, NQO1, TrxR, and Trx in PC12 cells were elevated remarkably by compounds 10 and 11 treatment in a dose
dependent manner, with HO-1 being promoted most notably, which is consistent with the genes expression level
result above. Accordingly, activities of NQO1 (Figure 10B), HO-1 (Figure 10C), TrxR (Figure 10D) and Trx
(Figure 10E) were all promoted to varying degrees in a dose-dependent manner. GCLC and GCLM, which are the
catalytic and regulatory subunits of the GSH biosynthesis enzyme GCL, form a heterodimer who catalyzes the
rate-limiting step in biosynthesis of GSH. GSH, a cardinal component of the glutathione system and a ubiquitous
cysteine-containing tripeptide antioxidant, can detoxify ROS and thereby play an important role in maintaining
intracellular redox homeostasis. As displayed in Figure 10A, total GSH level was elevated strikingly by treatment
of PC12 cells with 10 or 11. Collectively, compounds 10 or 11 showed great ability to upregulate the endogenous
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antioxidant species.
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Figure 8. Effect of compounds 10 and 11 on transcriptional expression of antioxidant genes and Nrf2. PC12 cells treated with (A) 10 and (B) 11 by 20
M were collected at indicated time and then the total RNA were extracted. Nrf2 and Phase II genes levels were normalized using GAPDH as an
internal control, and analyzed by RT-PCR. Representative graphs are from three independent experiments, with errors bars representing SD values. **P
< 0.01 vs the control group.
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Figure 9. Elevation of the content of corresponding antioxidant enzymes by compounds 10 and 11 in PC12 cells. (A and C) Promotion of the
expression of antioxidant enzymes determined by Western blots. After treatment with indicated concentrations of compounds 10 and 11 for 24 h, the
expression levels of HO-1, TrxR1, Trx1, and NQO1 in PC12 cells were determined. Quantification results of the Western blots are shown in B and D.
Representative graphs are from three independent experiments, with errors bars representing SD values. *P < 0.05 and **P < 0.01 vs the control group.
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Figure 10. Upregulation of activity or content of antioxidant species by 10 and 11 in PC12 cells. Elevation of (A) total GSH, (B) NQO1activity, (C)
HO-1 activity, (D) TrxR activity and (E) Trx activity by treatment of PC12 cells with indicated concentrations of 10 and 11. PC12 cells were treated
with indicated concentration of 10 and 11 for 24 h. Then antioxidant enzymes and GSH levels were determined. Representative graphs are from three
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independent experiments, with errors bars representing SD values. *P < 0.05 and **P < 0.01 vs the control group.
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77 2.6. Promotion of the Expression Level and Nuclear Translocation of Nrf2 by
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Compounds 10 and 11.
The translocation of Nrf2 from cytoplasm to nucleus is prerequisite for the Nrf2-dependent cytoprotective pathway,
which has attained broad consensus among scientists in this field. We then determined whether Nrf2 could
accumulate in nucleus by stimulation from compounds 10 and 11. After preparation of different fractions of PC12
cells treated by 10 and 11, western blots assays were employed to quantify the contents of Nrf2 in different parts of
the cells. As is displayed in Figure 11, the total Nrf2 was elevated slightly followed by 10 (Figure 11 A-B) or 11
(Figure 11 E-F) treatment, although the Nrf2 mRNA level was not influenced by these two compounds, indicating
that elevation of Nrf2 expression is conferred by post-transcription processes. Obviously, after 10 or 11 treatment,
cytoplasmic Nrf2 content declined gradually (Figure 11 A, D, E, and H), whereas the nuclear Nrf2 content
increased coincidently (Figure 11 A, C, E, and G), which indicates Nrf2 translocates to nucleus from cytoplasm.
Taken together, the results above demonstrate that 10 and 11 could promote both the nuclear translocation and
expression level of Nrf2, which would facilitate the binding of Nrf2 to ARE sequence so as to initiate the
transcription of antioxidant genes.
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Figure 11. Promotion of the level of Nrf2 expression and nuclear translocation of Nrf2 by compounds 10 or 11 treatment in PC12 cells. PC12 cells
were treated with 20 M compounds 10 or 11 for the indicated time, and then cells were collected and their fractions were prepared. (A and E) Western
blots analysis of total Nrf2, nuclear Nrf2, and cytosolic Nrf2. Quantification results of the Western blots are shown in B−D and F-H. Representative
graphs are from three independent experiments, with errors bars representing SD values. *P < 0.05 and **P < 0.01 vs the control group.
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We next assessed the contribution of Nrf2 for the preventive activity of 10 and 11. On this account, Nrf2
knockdown assay was conducted by transfecting PC12 cells with a shNrf2 plasmid specifically targeting Nrf2 to
generate stably transformed PC12 cells (PC12-shNrf2 cells). The control cells (PC12-shNT) were transfected with a
non-targeting shRNA plasmid. The transfection efficiency was examined by Western blot as shown in Figure 12A,
with the Nrf2 protein in PC12-shNrf2 cells decreased to 30% of the control PC12-shNT cells (Figure 12B). Then,
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we determined the preventive effect of 10 and 11 on oxidative stress-induced damages of both PC12-shNrf2 cells
and PC12-shNT cells. As shown in Figure 12C and Figure 12D, compounds 10 and 11 exerted their preventive
effects on PC12-shNT cells, while such preventive effects were almost entirely abrogated in PC12-shNrf2 cells,
indicating that Nrf2 is critically involved in the neuroprotection of PC12 cells by 10 and 11.
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Figure 12. Necessity of Nrf2 for cytoprotective activity of 10 and 11. (A, B) Determination of Nrf2 expression level in PC12-shNT and PC12-shNrf2
cells. PC12-shNT and PC12-shNrf2 cells were treated with indicated concentrations of 10 and 11 for 24 h, and then exposed to (C) 6-OHDA (200 M)
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or (D) H O (500 M) for 12 h. The cell viability was evaluated by the MTT assay. Representative graphs are from three independent experiments, with
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errors bars representing SD values. **P < 0.01 vs the control group; ^^P < 0.01 vs the 6-OHDA or H O treatment.
Continuous generation of ROS is a physiological outcome due to the aerobic metabolism of organisms, and indeed
ROS are essential to numerous physiological processes. However, once the ROS generation is beyond the control
of antioxidant defense mechnism, oxidative stress occurs, which results in lipid peroxidation, protein oxidation,
DNA damage, and ultimately cell death. PD is characterized by a progressive loss of dopaminergic neurons in the
substantia nigra pars compacta and the presence of intracellular protein deposition designated as Lewy bodies.
Although the molecular etiopathogenesis of PD is not fully understood, increasing evidences suggest that oxidative
stress acts as a critical role in the propagation stage of cellular damage during the development of PD. As a
consequence, a considerable amount of strategies to suppress oxidative stress are devoted to retarding or reversing
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the neurodegenerative progression.
Dithiolethiones have long been known to possess cancer chemopreventive activity that is ascribed to their ability to
induce phase 2 enzymes and antioxidant molecules by activation of Nrf2.40-42, 64 Additionally, several studies
demonstrated that certain dithiolethiones bearing different chemical structures had beneficial effects on
neurodegenerative diseases.44-48, 65 However, these sporadic studies limited the elucidation of the SAR of
dithiolethiones. In addition, the subtle mechanism of action is not well clarified in these studies. According to
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classifications of Nrf2 activators by Hu et al. , dithiolethiones are one of the important categories and are able to
arouse endogenous antioxidant defence mechanism of Nrf2-ARE. Under the frame of our ongoing researches
regarding the discovery and development of redox active small molecules as therapeutic or preventive agents,28,
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we described here the synthesis and evaluation of a series of structurally diverse dithiolethiones, and
discovered compounds 10 and 11 as the most potent neuroprotective agents to protect PC12 cells from the
6-OHDA- and H O -mediated neurotoxicity. The preliminary SAR of these dithiolethiones in preventing oxidative
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stress-induced damages of PC12 cells was summarized, which would guide further development and optimization
of such type of compounds. Although the exact mechanism by which dithiolethiones activate Keap1-Nrf2-ARE
pathway still remains to be elusive, the available research data demonstrate that at least three possibilities should be
considered. The hypersensitive sulfydryls of Keap1 could be covalently modified or oxidized or S-sulfhydrated by
dithiolethiones themselves and their metabolites. On the whole, these three mechanisms could be integrated as
covalent modification because of the alteration of the sulfydryls of the Keap1 protein. As firmly and elegantly
demonstrated by Kobayashi et al.61 and Smirnova et al. , D3T is able to react with Cys151 of Keap1 followed by
stabilization of Nrf2 and subsequent upregulation of antioxidant species. In this context, compounds 10 and 11 are
expected to exert their preventive effect in the similar way as D3T. Specifically, compounds 10 and 11 are
presumed to have an impact on cysteine-rich Keap1 protein by direct covalent modification of sulfydryl in Keap1
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and thereby disrupt the interaction between Keap1 and Nrf2, giving rise to the release of Nrf2 from cytoplasm to
migrating to nucleus where it stimulates the transcriptional activation of a panel of ARE-dependent cytoprotective
genes. These genes’ products then eliminate and detoxify the ROS and therefore confer the neuroprotection on
PC12 cells in combating with oxidative stress-induced damage. Genetic knockdown of Nrf2 expression almost
entirely abolishes the preventive effects of compounds 10 and 11, highlighting targeting Nrf2 is physiologically
important in exerting their neuroprotective effects in PC12 cells.
Regularly dietary intake of cruciferous vegetables, especially broccoli, could prevent our body from being damaged
by oxidative/electrophilic stress. However, it is worth mentioning that routine supplement of such vegetables is a
chemopreventive strategy against neurodegenerative diseases rather than a chemotherapeutic intervention tactics in
treatment of such disorders. Similarly, compounds 10 and 11 are thus postulated to exert their potent
chemopreventive effect in preventing the pathogenesis of neurodegenerative diseases or retarding the progression
of such disorders.
In summary, 59 structurally diverse dithiolethiones were synthesized and evaluated for neuroprotective activity in
PC12 cells, and compounds 10 and 11 were identified as the most active compounds, showing markedly better
preventive effects than famous D3T, CPDT and compound 2. Mechanistic studies demonstrated that activation of
the transcription factor Nrf2 is cardinally responsible for the neuroprotective effects of 10 and 11. Furthermore,
information obtained from SAR analysis would facilitate our further optimization of 10 and 11 to develop potential
neuroprotective candidates.
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59 4. Experimental Section
60 4.1. Chemistry
61 4.1.1. General Information
A Bruker AMX spectrometer was employed to obtain the H NMR and 13C NMR spectra using TMS as reference.
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The Shimadzu LCMS-2020 system or the Hewlett-Packard 5988A spectrometer is employed to analyze mass
spectra. All reagents and solvents were of reagent grade or purified according to standard methods before use.
Silica-gel plates (GF254) (Qingdao Haiyang Chemical Co., Ltd.) were used for monitoring the reaction under UV
light (254 nm). Silica gel (200~300 mesh) (Qingdao Haiyang Chemical Co., Ltd.) were used for column
chromatography.
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68 4.1.2. Compounds Purity Analysis
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The purity of all 59 final target compounds was determined by HPLC analysis, which was performed on Shimadzu
LC-20A system with a Wondasil C-18 Superb reversed-phase column (5 mm, 4.6 mm × 150 mm). Methanol and
water were used as mobile phase, and the flow rate is 0.6 mL/min. The tested compounds were dissolved in
chromatographic grade methanol, and the injection volume is 5 L. The detection wavelength in the range of
210~400 nm was employed to search for the maximal absorbance of the compounds, the purity of which
(compounds 1−59) is > 95% (RT stands for retention time).
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75 4.1.3. Synthesis of Intermediates
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Intermediates a1, b1, b2, c1, c2, d1, e1, e2, f1, f2, g1, h1, h2, h5, i1 and i3 were all obtained from commercially
available sources. Commercially available h1, h2 and i1 (70 mmol) were dissolved in anhydrous ethyl alcohol (180
mL) with injection of 2 mL of concentrated H SO . Then it was refluxed 12 hours and was directly evaporated to
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dryness. Then the residues were extracted dichloromethane and washed with large amount of water. Concentration
of combined organic phases to afford the crude intermediates h3, h4 and i2. These crude intermediates were used
for the next step without further purification. Intermediate acyl chloride i4 was obtained from the reaction of
benzoic acid i3 (8 mmol) in thionyl chloride (30 mL) as solvent under reflux for 3 h. Then the reaction was
evaporated under vacuum to obtain a colorless oil, which was reacted in the next step reaction directly. Key
intermediates c3 , f3 , f4 , h6 , h7 and i554 were synthesized according to the procedures described without
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any further modification.
Intermediates g2 were synthesized according to the method developed by Jae et al.52 with a little optimization. In
brief, the mixture of ethyl malonate potassium salt (2 eq), MgCl (1 eq) and TEA (2 eq) in anhydrous THF, which
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was stirred for 6 h at 25 C, was as A solution. The mixture of corresponding substituted benzoic acid (0.6 eq) and
1, 1’-carbonyldiimidazole (CDI) (0.8 eq) in anhydrous THF, which was refluxed to obtain a clear solution, was as
B solution. Then B was transferred to A, the mixture of which was refluxed overnight. The reaction was placed
under ice, and appropriate volume of 5 M HCl was added until pH 5~6. After extraction with EtOAc, workup as
described to provide the crude intermediates for further reaction without purification.
Ethyl 2-methyl-3-oxo-3-phenylpropanoate (h6-1) (R =CH ). Briefly, to an anhydrous THF solution of ethyl
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propionate (2 eq) under ice bath was added NaH (3 eq, 60% in mineral oil) portionwise. After 10 min, addition of
h3 (1 eq) was followed by refluxing overnight. The cooled reaction was directly poured into saturated aqueous
NH Cl, which was extracted using EtOAc. After concentration of the combined organic layer, it was purified with a
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short plug of silica gel through column chromatography. Yield: 92.3%; H NMR (400 MHz, CDCl ) δ: 8.003-7.974
3
(m, 2H), 7.615-7.572 (m, 1H), 7.505-7.467 (m, 2H), 4.382 (q, 1H, J=7.2 Hz, -CH(CH )-), 4.302 (q, 2H, J=7.2 Hz,
3
+
-OCH -), 1.501 (d, 3H, J=6.8 Hz, -CH ), 1.171 (t, 3H, J=7.2 Hz, -CH ); MS-ESI m/z: 229.0 [M+Na] .
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Ethyl 2-benzoylbutanoate (h6-2) (R =C H ). A similar method for h6-1 was employed to get compound h6-2
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exploiting ethyl butyrate and h3 as starting materials. Yield: 94.6%; H NMR (400 MHz, CDCl ) δ: 8.006-7.985 (m,
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2H), 7.613-7.571 (m, 1H), 7.504-7.466 (m, 2H), 4.239-4.124 (m, 3H, -CH(C H )-, -OCH -), 2.084-2.008 (m, 2H,
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+
-CH -), 1.176 (t, 3H, J=7.2 Hz, -CH ), 1.001 (t, 3H, J=7.2 Hz, -CH ); MS-ESI m/z: 243.0 [M+Na] .
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Ethyl 2-benzoyl-3-methylbutanoate (h6-3) (R =iso-propyl). A similar method for h6-1 was employed to get
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compound h6-3 exploiting ethyl isovalerate and h3 as starting materials. Yield: 66.4%; H NMR (400 MHz, CDCl )
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δ: 8.029 (d, 2H, J=7.2 Hz), 7.590 (t, 1H, J=7.6 Hz), 7.483 (t, 2H, J=7.6 Hz), 4.167-4.084 (m, 3H, -CH(C H )-,
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-OCH -), 2.705-2.615 (m, 1H, -CH(CH ) ), 1.182 (t, 3H, J=7.2 Hz, -CH ), 1.052 (d, 3H, J=6.4 Hz, -CH ), 0.943 (d,
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+
3H, J=6.4 Hz, -CH ); MS-ESI m/z: 257.0 [M+Na] .
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Ethyl 2-benzoylpentanoate (h6-4) (R =n-propyl). A similar method for h6-1 was employed to get compound h6-4
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exploiting ethyl valerate and h3 as starting materials. Yield: 75.6%; H NMR (400 MHz, CDCl ) δ: 7.997 (d, 2H,
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J=7.6 Hz), 7.590 (t, 1H, J=7.2 Hz), 7.485 (t, 2H, J=8.0 Hz), 4.310 (t, 1H, J=6.8 Hz, -CH(C H )-), 4.149 (q, 2H,
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J=7.2 Hz, -OCH -), 2.057-1.915 (m, 2H, -CH -), 1.435-1.343 (m, 2H, -CH ), 1.175 (t, 3H, J=7.2 Hz, -CH ), 0.953
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+
(t, 3H, J=7.6 Hz, -CH ); MS-ESI m/z: 257.0 [M+Na] .
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Ethyl 2-benzoylhexanoate (h6-5) (R =n-butyl). A similar method for h6-1 was employed to get compound h6-5
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exploiting ethyl hexanoate and h3 as starting materials. Yield: 80.3%; H NMR (400 MHz, CDCl ) δ: 8.009-7.984
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(m, 2H), 7.616-7.573 (m, 1H), 7.506-7.464 (m 2H), 4.290 (t, 1H, J=7.2 Hz, -CH(C H )-), 4.178-4.121 (m, 2H,
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-OCH -), 2.053-1.952 (m, 2H, -CH -), 1.397-1.305 (m, 4H, -CH -), 1.176 (t, 3H, J=6.8 Hz, -CH ), 0.897 (t, 3H,
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+
J=6.8 Hz, -CH ); MS-ESI m/z: 271.0 [M+Na] .
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Ethyl 2-benzoylheptanoate (h6-6) (R =n-pentyl). A similar method for h6-1 was employed to get compound h6-6
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1
exploiting ethyl heptanoate and h3 as starting materials. Yield: 77.8%; H NMR (400 MHz, CDCl ) δ: 8.003-7.981
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(m, 2H), 7.612-7.569 (m, 1H), 7.503-7.465 (m 2H), 4.288 (t, 1H, J=6.8 Hz, -CH(C H )-), 4.176-4.120 (m, 2H,
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-OCH -), 2.044-1.926 (m, 2H, -CH -), 1.359-1.290 (m, 6H, -CH -), 1.174 (t, 3H, J=7.2 Hz, -CH ), 0.871 (t, 3H,
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2
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3
+
J=6.8 Hz, -CH ); MS-ESI m/z: 285.0 [M+Na] .
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Ethyl 2-benzoyloctanoate (h6-7) (R =n-hexyl). A similar method for h6-1 was employed to get compound h6-7
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1
exploiting ethyl caprylate and h3 as starting materials. Yield: 74.5%; H NMR (400 MHz, CDCl ) δ: 8.003-7.981
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(m, 2H), 7.614-7.571 (m, 1H), 7.509-7.466 (m 2H), 4.288 (t, 1H, J=6.8 Hz, -CH(C H )-), 4.176-4.119 (m, 2H,
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-OCH -), 2.029-1.962 (m, 2H, -CH -), 1.351-1.252 (m, 8H, -CH -), 1.174 (t, 3H, J=7.2 Hz, -CH ), 0.865 (t, 3H,
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J=7.2 Hz, -CH ); MS-ESI m/z: 277.1 [M+H] .
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Ethyl 2-methyl-3-oxo-3-(3,4,5-trimethoxyphenyl)propanoate (h7-1) (R =CH ). A similar method for h6-1 was
3 3
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employed to get compound h7-1 exploiting ethyl propionate and h4 as starting materials. Yield: 88.9%; H NMR
(400 MHz, CDCl ) δ: 7.268 (s, 2H), 4.382 (q, 1H, J=7.2 Hz, -CH(CH )-), 4.302 (q, 2H, J=7.2 Hz, -OCH -),
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2
3.931-3.906 (m, 9H, -OCH ), 1.502 (d, 3H, J=6.8 Hz, -CH ), 1.203 (t, 3H, J=7.2 Hz, -CH ); MS-ESI m/z: 297.0
3
3
3
[M+H]+.
Ethyl 2-(3,4,5-trimethoxybenzoyl)butanoate (h7-2) (R =C H ). A similar method for h6-1 was employed to get
3
2
5
1
compound h7-2 exploiting ethyl butyrate and h4 as starting materials. Yield: 91.4%; H NMR (400 MHz, CDCl ) δ:
3
7.286 (s, 2H), 4.190-4.137 (m, 3H, -CH(C H )-, -OCH -), 3.929 (s, 3H, -OCH ), 3.924 (s, 6H, -OCH ), 2.086-2.013
2
5
2
3
3
+
(m, 2H, -CH -), 1.205 (d, 3H, J=7.2 Hz, -CH ), 1.002 (t, 3H, J=7.2 Hz, -CH ); MS-ESI m/z: 311.1 [M+H] .
2
3
3
Ethyl 3-methyl-2-(3,4,5-trimethoxybenzoyl)butanoate (h7-3) (R =iso-propyl). A similar method for h6-1 was
3
1
employed to get compound h7-3 exploiting ethyl isovalerate and h4 as starting materials. Yield: 55.7%; H NMR
(400 MHz, CDCl ) δ: 7.319 (s, 2H), 4.193-4.095 (m, 2H, -OCH -), 4.012 (d, 1H, J=9.6 Hz, -CH(C H )-), 3.931 (s,
3
2
3
7
9H, -OCH ), 2.733-2.609 (m, 1H, -CH(CH ) ), 1.212 (t, 3H, J=7.2 Hz, -CH ), 1.052 (d, 3H, J=6.8 Hz, -CH ), 0.951
3
3 2
3
3
+
(d, 3H, J=6.8 Hz, -CH ); MS-ESI m/z: 325.0 [M+H] .
3
Ethyl 2-(3,4,5-trimethoxybenzoyl)pentanoate (h7-4) (R =n-propyl). A similar method for h6-1 was employed to
3
1
get compound h7-4 exploiting ethyl valerate and h4 as starting materials. Yield: 80.6%; H NMR (400 MHz,
CDCl ) δ: 7.281 (s, 2H), 4.255 (t, 1H, J=7.2 Hz, -CH(C H )-), 4.162 (q, 2H, J=7.2 Hz, -OCH -), 3.930 (s, 3H,
3
3
7
2
-OCH ), 3.923 (s, 6H, -OCH ), 2.061-1.923 (m, 2H, -CH -), 1.434-1.340 (m, 2H, -CH ), 1.205 (t, 3H, J=7.2 Hz,
3
3
2
3
+
-CH ), 0.969 (t, 3H, J=7.2 Hz, -CH ); MS-ESI m/z: 325.0 [M+H] .
3
3
Ethyl 2-(3,4,5-trimethoxybenzoyl)hexanoate (h7-5) (R =n-butyl). A similar method for h6-1 was employed to get
3
1
compound h7-5 exploiting ethyl hexanoate and h4 as starting materials. Yield: 82.3%; H NMR (400 MHz, CDCl )
3
δ: 7.282 (s, 2H), 4.233 (t, 1H, J=7.2 Hz, -CH(C H )-), 4.162 (q, 2H, J=7.2 Hz, -OCH -), 3.930 (s, 3H, -OCH ),
4
9
2
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3.923 (s, 6H, -OCH ), 2.035-1.980 (m, 2H, -CH -), 1.410-1.311 (m, 4H, -CH -), 1.204 (t, 3H, J=7.2 Hz, -CH ),
3 2 2 3
+
0.909 (t, 3H, J=6.8 Hz, -CH ); MS-ESI m/z: 339.0 [M+H] .
3
Ethyl 2-(3,4,5-trimethoxybenzoyl)heptanoate (h7-6) (R =n-pentyl). A similar method for h6-1 was employed to
3
1
get compound h7-6 exploiting ethyl heptanoate and h4 as starting materials. Yield: 74.2%; H NMR (400 MHz,
CDCl ) δ: 7.281 (s, 2H), 4.234 (t, 1H, J=7.2 Hz, -CH(C H )-), 4.162 (q, 2H, J=7.2 Hz, -OCH -), 3.929 (s, 3H,
3
5
11
2
-OCH ), 3.922 (s, 6H, -OCH ), 2.030-1.970 (m, 2H, -CH -), 1.377-1.325 (m, 6H, -CH -), 1.204 (t, 3H, J=7.2 Hz,
3
3
2
2
+
-CH ), 0.897-0.864 (m, 3H, -CH ); MS-ESI m/z: 353.0 [M+H] .
3
3
Ethyl 2-(3,4,5-trimethoxybenzoyl)octanoate (h7-7) (R =n-hexyl). A similar method for h6-1 was employed to get
3
1
compound h7-7 exploiting ethyl caprylate and h4 as starting materials. Yield: 58.6%; H NMR (400 MHz, CDCl )
3
δ: 7.280 (s, 2H), 4.234 (t, 1H, J=7.2 Hz, -CH(C H )-), 4.161 (q, 2H, J=7.2 Hz, -OCH -), 3.930 (s, 3H, -OCH ),
6
13
2
3
3.923 (s, 6H, -OCH ), 2.031-1.974 (m, 2H, -CH -), 1.361-1.187 (m, 11H, -CH -, -CH ), 0.891-0.857 (m, 3H, -CH );
3
2
2
3
3
MS-ESI m/z: 389.1 [M+Na]+.
4
4
4
4
4
4
4
4
4
4
63 4.1.4. Target Compounds Synthesis and Characterization
64
65
66
67
68
69
70
71
72
Compound 1 and compound 6 were synthesized by the methods invented by Behringer et al.55 and Jin et al.56
without any modification, respectively. Except for compounds 1 and 6, the other target compounds were
synthesized in the light of the procedure developed by Curphey et al.57 with a little optimization. In brief, ~20 mL
anhydrous xylene containing various intermediates g2 (5 mmol) was injected dropwise into the suspension of the
P S (3 mmol), HMDO (15 mmol), and sulfur (5 mmol) in 30 mL of anhydrous xylene under reflux. The refluxed
4
10
reaction was terminated until no original materials g2 were observed monitored by TLC. Addition of K CO3
2
o
solution (6.68 mmol K CO /mmol of P S ) was conducted at 0 C. Then addition of ~25 mL acetone (equal to half
2
3
4 10
of the reaction solvent) was followed by stirring for thirty min at 0 °C. Extraction of the obtained maroon material
with toluene was followed by purification through a short column of activated carbon (~4 g) to obtain a clear
2
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maroon solution, which was concentrated and purified through column chromatography.
Because of inaccessibility of pure g3 and g4 as target compounds, the crude g3 and g4 were directly reduced
according to Reddy et al.67 in order to provide target compounds 42 and 43. In the same way, 41 was obtained by
reduction of 38.
3H-1,2-dithiole-3-thione (Compound 1). Compound 1 was synthesized according to a published procedure55 to
1
give a khaki solid. Yield: 69.6%; H NMR (400 MHz, DMSO-d ) δ: 8.980 (d, 1H, J=5.6 Hz), 7.296 (d, 1H, J=5.6
6
Hz); 13C NMR (100 MHz, DMSO-d ) δ: 216.703, 160.307, 139.686; MS-ESI m/z: 135.00 [M+H] ; Purity
+
6
determination: RT=4.28 min, purity 99.90%, 80:20 CH OH/H O.
3
2
Methyl 3-thioxo-3H-1,2-dithiole-4-carboxylate (Compound 2). A published method39 was employed to afford
1
Compound 2 as a red solid. Yield: 47.2%; H NMR (400 MHz, CDCl ) δ: 9.206 (s, 1H), 3.897 (s, 3H, -COOMe);
3
1
3
+
C NMR (100 MHz, CDCl ) δ: 211.164, 165.548, 161.210, 137.959, 52.853; MS-ESI m/z: 193.00 [M+H] ; Purity
3
determination: RT=3.96 min, purity 98.53%, 80:20 CH OH/H O.
3
2
5,6-dihydrocyclopenta[c][1,2]dithiole-3(4H)-thione (Compound 3). Compound 3 was obtained from b1 as
starting material following the general procedure described by Curphey et al.57 to provide a khaki solid. Yield:
66.7%; H NMR (400 MHz, CDCl ) δ: 2.970-2.934 (m, 2H), 2.698-2.571 (m, 4H); 13C NMR (100 MHz, DMSO-d6)
1
3
+
δ: 208.088, 175.347, 155.680, 34.089, 30.435, 29.011; MS-ESI m/z 175.05 [M+H] ; Purity determination: RT=5.30
min, purity 99.75%, 85:15 CH OH/H O.
3
2
4,5,6,7-tetrahydro-3H-benzo[c][1,2]dithiole-3-thione (Compound 4). A similar method for compound 3 was
1
employed to get compound 4 exploiting b2 as starting material to afford a light khaki solid. Yield: 49.2%; H NMR
(400 MHz, CDCl ) δ: 2.817 (t, 2H, J=5.2 Hz), 2.581 (t, 2H, J=6.0 Hz), 1.860-1.767 (m, 4H); 13C NMR (100 MHz,
3
+
CDCl ) δ: 215.311, 169.343, 143.390, 29.653, 27.522, 21.381, 20.806; MS-ESI m/z: 189.00 [M+H] ; Purity
3
determination: RT=6.43 min, purity 99.92%, 85:15 CH OH/H O.
3
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5-methyl-3H-1,2-dithiole-3-thione (Compound 5). A similar method for compound 3 was employed to get
1
compound 5 exploiting e1 as starting material to afford a maroon oil. Yield: 81.3%; H NMR (400 MHz, DMSO-d )
6
δ: 7.214 (s, 1H), 2.523 (d, 3H, J=0.8 Hz, -CH ); 13C NMR (100 MHz, DMSO-d ) δ: 215.665, 175.420, 138.955,
3
6
+
17.826; MS-ESI m/z: 149.00 [M+H] ; Purity determination: RT=4.40 min, purity 99.93%, 85:15 CH OH/H O.
3
2
3H-benzo[c][1,2]dithiole-3-thione (Compound 6). Compound 6 was prepared from d1 as starting material
following method developed by Jin et al.56 to provide an orange solid. Yield: 74.5%; H NMR (400 MHz, CDCl ) δ:
1
3
8.225 (d, 1H, J=8.0 Hz), 7.750-7.667 (m, 2H), 7.485-7.445 (m, 1H); 13C NMR (100 MHz, CDCl ) δ: 215.563,
3
+
173.073, 135.973, 132.281, 131.622, 129.689, 126.959; MS-ESI m/z: 185.00 [M+H] ; Purity determination:
RT=9.75 min, purity 99.92%, 80:20 CH OH/H O.
3
2
5-phenyl-3H-1,2-dithiole-3-thione (Compound 7). A similar method for compound 3 was employed to get
1
compound 7 exploiting e2 as starting material to afford a metallic shiny yellow solid. Yield: 80.9%; H NMR (400
MHz, CDCl ) δ: 7.684-7.655 (m, 2H), 7.590-7.547 (m, 1H), 7.519-7.474 (m, 2H), 7.452 (s, 1H); 13C NMR (100
3
+
MHz, CDCl ) δ: 216.812, 152.856, 140.785, 132.804, 128.571, 126.089, 124.398; MS-ESI m/z: 211.00 [M+H] ;
3
Purity determination: RT=8.57 min, purity 99.56%, 80:20 CH OH/H O.
3
2
Indeno[1,2-c][1,2]dithiole-3(4H)-thione (Compound 8). A similar method for compound 3 was employed to get
1
compound 8 exploiting f3 as intermediate to afford a khaki solid. Yield: 37.5%; H NMR (400 MHz, DMSO-d ) δ:
6
8.033 (d, 1H, J=7.6 Hz), 7.670 (d, 1H, J=7.6 Hz), 7.576 (t, 1H, J=7.6 Hz), 7.493 (t, 1H, J=7.2 Hz), 3.746 (s, 2H);
1
3
C NMR (100 MHz, DMSO-d ) δ: 205.638, 171.415, 152.826, 148.981, 137.381, 130.581, 127.858, 126.311,
6
+
122.529, 34.949; MS-ESI m/z: 223.00 [M+H] ; Purity determination: RT=9.86 min, purity 99.02%, 80:20
CH OH/H O.
3
2
4,5-dihydro-3H-naphtho[1,2-c][1,2]dithiole-3-thione (Compound 9). A similar method for compound 3 was
1
employed to get compound 9 exploiting f4 as intermediate to afford a red solid. Yield: 86.8%; H NMR (400 MHz,
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