The synthesis of sulforaphane analogues and their protection effect against cisplatin induced cytotoxicity in kidney cells

Article abstract of DOI:10.1016/j.bmcl.2014.11.014

A series of sulforaphane analogues were synthesized with various amines by treatment of carbon disulfide followed by Boc₂O and DMAP. These synthesized sulforaphane analogues were tested on cisplatin treated cultured LLC-PK1 kidney cell line. Among these analogues, several compounds including SF5 show a potent effect on kidney cell protection assay at the concentration of 2.5 μM. Further studies with compound SF5 revealed that the kidney cell protection effect was related by inhibiting the apoptosis pathway through JNK-p53-caspase apoptotic cascade. Compound SF5 may be considered as a promising candidate for the development of new kidney protection agent against drug induced acute kidney disease.

Full text of DOI:10.1016/j.bmcl.2014.11.014

Bioorganic & Medicinal Chemistry Letters 25 (2015) 62–66
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis of sulforaphane analogues and their protection effect
against cisplatin induced cytotoxicity in kidney cells
Taejung Kim a, Young-Joo Kim a, Im-Ho Han a, Dahae Lee a,b, Jungyeob Ham a, Ki Sung Kang b,
,
⇑
Jae Wook Lee a,c,
⇑
a Natural Product Research Center, Korea Institute of Science and Technology, Gangneung 210-340, Republic of Korea
b College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea
c Department of Biological Chemistry, University of Science and Technology, Daejeon 305-350, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 July 2014
Revised 17 October 2014
Accepted 5 November 2014
Available online 11 November 2014
A series of sulforaphane analogues were synthesized with various amines by treatment of carbon disul-
fide followed by Boc2O and DMAP. These synthesized sulforaphane analogues were tested on cisplatin
treated cultured LLC-PK1 kidney cell line. Among these analogues, several compounds including SF5
show a potent effect on kidney cell protection assay at the concentration of 2.5 lM. Further studies with
compound SF5 revealed that the kidney cell protection effect was related by inhibiting the apoptosis
pathway through JNK-p53-caspase apoptotic cascade. Compound SF5 may be considered as a promising
candidate for the development of new kidney protection agent against drug induced acute kidney
disease.
Keywords:
Sulforaphane
Cisplatin induced kidney disease
Kidney protection
Reactive oxygen species
Isothiocyanate
Ó 2014 Elsevier Ltd. All rights reserved.
The increasing incidence of drug-induce acute kidney disease
has led to a societal interest in development of new therapeutic
strategies to overcome this condition. Kidney disease related to
side effect of drug is considered as a limitation to the effective
treatment for many patients. In particular, cisplatin is a powerful
anticancer chemotherapeutic agent which can dramatically
improve the survival rate of cancer patients. Although cisplatin
has been used as mainstay of cancer therapy for many years, severe
side effects of cisplatin, particularly nephrotoxicity, limits its wide
spread use for cancer patients.1 For many years, various
approaches have been taken to curtail the side effect of cisplatin.
However, nephrotoxicity still remains a major concern associated
with cisplatin based cancer therapy. The mechanism of cisplatin
nephrotoxicity is related to various cell signal pathways including
cell death promoting apoptosis,2 MAPK,3 p53,4 and reactive oxygen
or rats.8 Therefore, there is no verifiable treatment for patients to
prevent cisplatin induced nephrotoxicity except for kidney
dialysis.
Recently, an appropriate treatment to reduce the risk of these
kidney failures is offered by the identification of chemoprotective
agents from natural sources. Chemoprotection by natural products
increases the cellular major defense mechanism by cells against
endogenous and environmental attack resulting from ROS and
electrophilic species.9 The natural chemoprotective agent sulfora-
phane, a naturally occurring isothiocyanate derived from glucosin-
olate, is present in cruciferous vegetable such as broccoli, brussels
sprouts or cabbages.5 Sulforaphane has gained attention as a che-
moprotective agent which can enhance the expression of classical
phase 2 antioxidant enzymes and cytoprotective proteins includ-
ing NAD(P)H:quinone oxidoreductase (NQO1),10 thioredoxin,
superoxide dismutase (SOD), heme oxidase-1 (HO-1), glutathione
peroxidase and glutathione S-transferase (GST).11 Extensive
research in vitro and in vivo has already demonstrated the poten-
tial of sulforaphane to protect or reduce against cancer,12 skin
damage,13 and renal damage14 resulting from reduce oxidative
stress,11a,12a and UV irradiation.15
species (ROS)5 or cytoprotective p21.6 Cisplatin also induces TNF-
a
production in tubular cells that triggers an inflammatory response7
and cell injury and death. There are several pharmacological
approaches to address the problem such as using CDK, p53, MAPK
inhibitors, antioxidant, and anti-inflammation to protect kidney
cell during cisplatin treatment. Yet, most studies of the effects
and treatments have been conducted only in cultured cells, mice,
In our prior study, sulforaphane is identified as a potent
Renoprotective agent for cultured LLC-PK1 cells under cisplatin
induced oxidative stress. To discover more potent sulforaphane
analogues, we conducted structure activity relationship (SAR)
studies by considering on the characteristics of side chain such
⇑
Corresponding authors. Tel.: +82 31 750 5402; fax: +82 31 750 5416 (K.S.K.);
tel.: +82 33 650 3514; fax: +82 33 650 3529 (J.W.L.).
E-mail addresses: kkang@gachon.ac.kr (K.S. Kang), jwlee5@kist.re.kr (J.W. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.11.014
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

T. Kim et al. / Bioorg. Med. Chem. Lett. 25 (2015) 62–66
63
as lipophilicity, chain length, and steric factor. In the previous
studies, the characteristic of side chain of isothiocyanate (ITC)
influence not only lipophilicity, but also the reactivity of the
isothiocyanate moiety.16 Therefore, we designed 16 aliphatic
ITCs or aromatic ITCs. To generate 16 sulforaphane analogues,
we followed the previously reported procedure describe below
(Fig. 1).16a
At first, various sulforaphane analogues were prepared from
various aliphatic and aromatic amines (1) reacted with carbon
disulfide under basic condition followed by the addition of di-
tert-butyl dicarbonate (Boc2O) and 4-dimethylaminopyridine
(DMAP). These synthesized aliphatic and aromatic sulforaphane
analogues were purified by silica gel column chromatography.
The chemical structure of synthesized aliphatic and aromatic sulfo-
raphane analogues were characterized by 1H NMR, 13C NMR, and
LC–MS.
80% of cell survival at 2.5
l
M concentration, which is more potent
than sulforaphane. SF5 show strong cell survival effect of kidney
epithelial cells (93% at a concentration of 2.5 M). Analogues
SF12 also show 90% cell survival at a concentration of 10 M,
which is similar or slightly better than sulforaphane. 1-Phenyle-
thane group (SF7) and methoxyphenyl group (SF9) led to a
decrease in activity due to their steric effect. In contrast, chloro-
benzyl (SF6), hydroxyphenethyl (SF8), and diphenethyl group
(SF5) appear to be tolerated and increase activity. Based on the
SAR, we selected SF517 for further study considering a cellular
potency (Fig. 2).
To confirm the potency of SF5 to cell survival effect, we
observed the image of 25 lM of cisplatin treated LLC-PK1 cell fol-
lowing treatment with different concentration of SF5. In the cell
image data, cisplatin treated LLC-PK1 cell showed growth arrest
or cell death. However, LLC-PK1 cells treated with different con-
l
l
To evaluate molecules that enhance cell survival, we utilized a
previously developed cell-based assay with UV–vis readout. This
cell-based assay format is commonly used for the identification
of molecules that enhance cell-survival. In LLC-PK1 cell-based
centration of SF5 showed improved cell survival above 1 lM con-
centration of SF5. We confirmed that SF5 has a cytoprotective
effect to cisplatin treated LLC-PK1 cells (Fig. 3).
To further evaluate the cytoprotective effects of SF5, we per-
formed the image based cytometric assay.18 Figure 4 shows the
effects of SF5 on apoptosis in LLC-PK1 cells. As shown in Figure 4A,
the number of dead and apoptotic cells which were stained
with red and green color were increased after cisplatin treatment,
whereas it was decreased by the co-treatment of SF5. The
elevated percentage of apoptotic cells by cisplatin treatment in
LLC-PK1 cells was markedly decreased by the co-treatment of
SF5 (Fig. 4B).
To investigate the mechanism of protection effect of SF5 in LLC-
PK1 cells, we analyzed expression of JNK, p53, and caspase-3 by
western blot analysis. In western blot analysis, cisplatin induced
high levels of phosphorylated JNK (phospho-JNK) and cleavage of
caspase-3, which activated apoptosis of LLC-PK1 cells. While differ-
ent concentration of SF5 shows reduction of phospho-JNK, p53, and
cleaved caspase-3 protein levels.
assay condition, cisplatin (25 lM) is treated to generate oxidative
stress on LLC-PK1 cells. The oxidative stress activates cell death
signal pathway, which results in 60% of cell survival. Using this
condition, 16 sulforaphane analogues were tested in a dose
response format in the LLC-PK1 cell-based assay. We then evalu-
ated cell survival rate by WST-1 assay using cell counting kit
(CCK-8 kit).
We defined the potency of the compounds by calculating the
concentration which resulted in more than 60% cell survival. Sulfo-
raphane (10 lM) increase more than 77% of cell survival with min-
imal effect on cell toxicity in LLC-PK1 cells. Most of sulforaphane
analogues showed similar or better potency compared with sulfo-
raphane in cell survival assay. Effects of sulforaphane analogues
were tested up to 10
lM, whereas toxic analogues at high doses
were tested up to 2.5
l
M. SF6, SF8 and SF13 show more than
Figure 1. The synthetic scheme of sulforaphane and chemical structure of sulforaphane analogues.

64
T. Kim et al. / Bioorg. Med. Chem. Lett. 25 (2015) 62–66
Figure 2. LLC-PK1 protection assay of sulforaphane and sulforaphane analogues under the treatment of 25 l
M of cisplatin. *p < 0.05 compared to the cisplatin-treated value.
Earlier studies showed that the cisplatin-induced damages in
LLC-PK1 cells was largely attenuated by the antioxidant N-acetyl-
cysteine, while apoptosis was prevented by the specific inhibitors
for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-alpha but
not by the p38 MAPK inhibitor SB203580.19 JNK is another
important MAPK which is involved in the cytoprotective effect of
selenium on cadmium-induced oxidative renal cell damage.20
In the present study, we sought to determine the role of the
JNK-p53-caspase-3 apoptotic cascade in mediating the protective
effect of SF5 against oxidative cytotoxicity in renal cells. As shown
in Figure 5, phosphorylation of JNK was observed at 24 h after
cisplatin treatment, and it was decreased after treatment with
SF5 in a dose dependent manner. In Figure 5, protein level of p53
was also markedly increased after treatment of cisplatin, whereas
the elevated level of p53 was reduced significantly by a high con-
centration (2.5 lM) of SF5. Similarly, the elevated protein level of
cleaved caspase-3 also decreased after treatment with SF5 in a
dose dependent manner.
In summary, the results of our present study show that the
JNK-p53-caspase-3 signaling cascade plays a critical role in medi-
ating the protective effect of SF5 against oxidative cytotoxicity in
cultured LLC-PK1 cells.

T. Kim et al. / Bioorg. Med. Chem. Lett. 25 (2015) 62–66
65
Figure 3. The image of nephroprotection effect with SF5 under the treatment of 25 lM of cisplatin.
Figure 4. Effects of SF5 on apoptosis in LLC-PK1 cells. (A) Representative images for apoptosis detection. (B) Percentage of Annexin V-positive-stained apoptotic cells. Dead
cell and apoptotic cells were stained with red and green color. Apoptosis was determined by a Tali image-based cytometer.
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion of final compounds and biological assay protocols) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2014.11.014.
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