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D. A. Brown et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5829–5831
Figure 1. Structures of dithiolethiones.
with molecular oxygen to generate superoxide anion, OÅ2À, capable
of dismutating into hydrogen peroxide, H2O2, which affects a disul-
fide closure on Keap1.19–22 Indeed, the induction of NQO1, a highly
inducible phase 2 enzyme, by ADT and D3T can be abrogated by
catalase, supporting the role of hydrogen peroxide in Nrf2
activation.18
The full potential of these remarkable molecules as neuropro-
tective agents is unknown, as limited studies have been performed
in neuronal models.11,12,23,24 One aim of our research is to explore
the therapeutic potential of dithiolethiones as disease-modifying
antiparkinsonian agents. Towards this, we herein describe the syn-
thesis, GSH induction, SAR, and neuroprotective properties of a ser-
ies of structurally-diverse dithiolethiones in the SH-SY5Y
neuroblastoma cell line, commonly used in in vitro PD models.
Our study commenced with the synthesis of substituted dithiol-
ethiones, depicted in Scheme 1 and Table 1. We chose to use this
set of molecules in our initial SAR study because structural diver-
sity was desired. Appropriately substituted b-oxo esters or b-keto
esters 1a–h reacted smoothly with elemental sulfur and Lawes-
son’s reagent in toluene for 6 h to give the titled dithiolethiones
2a–h in 39–75% yield.25,26
Synthesized dithiolethiones 2a–h, along with commercially
available dithiolethiones in Figure 1, were assayed for glutathione
induction in SH-SY5Y cells at concentrations of 100 lM after 24 h.
Total cellular glutathione (GSH and GSSG) was measured using the
GSH/GSSG GloÓ assay from Promega. The results are shown in
Figure 2 and are reported as a percentage of control.
Of the molecules evaluated, D3T, CPDT, and 2d significantly
increased total GSH levels by 215%, 183%, and 175%, respectively.
Surprisingly, the simplest of the dithiolethiones, D3T, increased
glutathione the most. Amongst the 5-monosubstituted com-
pounds, those possessing an electron-donating alkyl group (2a,
2b, and 2d) were more potent glutathione inducers than those con-
taining a phenyl ring (ADT, ADTOH, and 2c). Interestingly, replace-
ment of an electron donating group on the phenyl ring (ADT and
Figure 2. Total glutathione levels. Compounds were evaluated at a concentration of
100 M. Data shown are mean SEM of at least three different experiments.
⁄P < 0.005.
l
ADTOH) with one of electron-withdrawing character (2c) abol-
ished activity. Various 4-monosubstituted molecules demon-
strated similar potencies; however, an electron withdrawing
group at this position was slightly favored (2e, 2g, and 2h vs 2f
and 2h). Of the bicyclic dithiolethiones tested, cyclopenta CPDT
strongly induced glutathione (183%), whereas the cyclohexa
homolog, CHDT, was inactive (98%). These data, taken together,
suggest that placement of an electron-donating alkyl group at
the 5-position and an electron-withdrawing group at the 4-posi-
tion, whether alone or in combination (2d), may be optimal for
induction of GSH in substituted dithiolethiones.
The differences in glutathione induction activity in the evalu-
ated molecules may be related to their susceptibility to reduction,
as various dithiolethiones have been observed to undergo thiol-
mediated reduction at different rates.27 As such, the electronic
properties of dithiolethiones are likely to govern their modulation
of Keap1, as described above. However, as D3T was the best indu-
cer of GSH, additional factors may also play a role in this process,
including sterics and production of active metabolites. It is also
conceivable D3T may increase GSH levels by multiple mechanisms,
such as activation of AMP-activated protein kinase (AMPK).28 Addi-
tional SAR studies and pharmacological investigation will provide
more information regarding the relationship between dithiolethi-
ones and GSH induction.
Scheme 1. Synthesis of substituted dithiolethiones.
As GSH induction is a result of Nrf2-mediated transcription of
cytoprotective genes, we next explored abilities of dithiolethiones
D3T, CPDT, 2c, and 2d to protect SH-SY5Y cells against 6-hydroxy-
dopamine (6-OH DA) induced neurotoxicity, a routinely employed
cell culture PD model.29–32 D3T, CPDT, and 2d were evaluated as
they caused a significant increase in GSH, and 2c since no GSH
induction was observed. SH-SY5Y cells were pretreated with test
Table 1
Structures of synthesized dithiolethiones
Entry
R1
R2
Entry
Yield (%)
1a
1b
1c
1d
Ethyl
Cyclopropyl
p-Chlorophenyl
Methyl
H
H
H
H
H
H
H
Cl
2a
2b
2c
2d
2e
2f
53
39
71
58
63
75
43
44
compounds (100
50 M) for an additional 24 h, at which time cell viability was
assessed using CellTiter GloÓ assay from Promega. As shown in
Figure 3, treatment of SH-SY5Y cells with 25 and 50 M 6-OH DA
decreased cell viability to 23% and 14% of control, respectively.
lM) for 24 h, then exposed to 6-OH DA (25 and
1e25
1f25
1g25
1h25
p-Methoxyphenyl
p-Chlorophenyl
Phenyl
l
2g
2h
l
1,1,1-Trifluoroethyl