Inhibition of glutathione S-Transferase M1 by new gabosine analogues is essential for overcoming cisplatin resistance in lung cancer cells

Article abstract of DOI:10.1021/jm201131n

A new class of human GST inhibitors has been identified via rational design approach; we report their discovery, synthesis, inhibitory activity, and synergetic effect in combination with cisplatin against A549 lung cancer cell line. The results of this ef

Full text of DOI:10.1021/jm201131n

Article
pubs.acs.org/jmc
Inhibition of Glutathione S-Transferase M1 by New Gabosine
Analogues Is Essential for Overcoming Cisplatin Resistance in Lung
Cancer Cells
Chie-Hong Wang,† Ho T. Wu,‡ Hau M. Cheng,‡ Tien-Jui Yen,† I-Hsuan Lu,† Hui Chuan Chang,†
Shu-Chuan Jao,§ Tony K. M. Shing,*,‡ and Wen-Shan Li*,†,∥
†Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
‡Department of Chemistry and Center of Novel Functional Molecules, The Chinese University of Hong Kong, Shatin, Hong Kong,
China
§Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
∥Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
S
* Supporting Information
ABSTRACT: A new class of human GST inhibitors has been identified via
rational design approach; we report their discovery, synthesis, inhibitory
activity, and synergetic effect in combination with cisplatin against A549
lung cancer cell line. The results of this effort show that the lead 4-O-decyl-
gabosine D (24) has optimum synergetic effect in A549 human lung
adenocarcinoma epithelial cell and that this activity involves inhibition of
glutathione S-transferase M1, apparently consistent with siRNA-mediated
knockdown of GSTM1 gene.
INTRODUCTION
■
Human glutathione S-transferases (hGSTs), a family of GSH-
dependent enzymes in phase II detoxification system,
participate in drug resistance processes by catalyzing the con-
jugation of glutathione (GSH) to a wide variety of anticancer
drugs (e.g., cisplatin, oxaliplatin, thiotepa, chlorambucil,
melphalan, cyclophosphamide, doxorubicin, and so on).1 The
resulting GS−drug conjugates are more hydrophilic and are
eliminated from the cell through transporter proteins, multi-
drug-resistance-related proteins (MRP) and P-glycoprotein
(P-gp).2 This operating mechanism elevates the efflux activity
of drugs in cancer cells, resulting in anticancer drug resistance
(Figure 1).3 To date, traditional GST inhibitors are customarily
considered to be used with anticancer drugs for cancer patients
to prevent multidrug resistance occurrence. Consequently,
investigations and development on GST inhibitors would have
practical applications in chemoresistance.
Figure 1. Possible GST-mediated activation of anticancer drug
resistance in lung cancer cells.
Eight classes of GSTs in mammals have been identified on
the basis of their biofunctional properties and sequence
identities.4 Among these GST isoenzymes, GST P1-1, GST
A2, and GST M1 are overexpressed in many cancer cell lines
including breast, lung, and ovarian cancers.5 There are two
main types of GST inhibitors: non-GSH compounds6 and GSH
analogues.7 Although diverse GST inhibitors have been
identified by several distinctive strategies, they are classically
restricted to GSH−ethacrynic acid derivatives with poor cell
membrane permeability. Gabosines, a family of multihydroxy-
lated cyclohexenones and cyclohexanones,8 have been developed
Received: August 24, 2011
Published: November 15, 2011
© 2011 American Chemical Society
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and synthesized previously in our group.9 In particular, some
gabosines have been demonstrated to exhibit biological
applications such as antibiotics, anticancer agents, and DNA
intercalating agents,10 thereby inspiring us to pursue research
on the capability of gabosine analogues on GST inhibition.
Herein we report a rapid discovery of new GST inhibitors via
rational design strategy and identify the capability of 4-O-decyl-
gabosine D (24) to suppress the resistance against cisplatin in
lung cancer cells by inhibiting GSTM1.
reaction of a diketone derived from D-glucose.9c Deacetonation
of 4 under mild acid hydrolysis gave diol 5, which was regio-
selectively esterified to yield acetate 6. Enone 4 was stereo-
selectively reduced by sodium borohydride or K-selectride to
give known β-allylic alcohol 259c or α-allylic alcohol 26,9b
respectively. Alkylation of the free alcohol in 25 and 26
afforded the corresponding ethers 27 and 28, which were
converted into respective enones 10, 11, and 15−23 in three
steps involving deacetonation, regioselective acetylation, and
pyridinium dichromate (PDC) oxidation. Acid hydrolysis of the
trans-diacetal goup in 10, 11, 15, 16, and 23 furnished enone-
diols 7, 8, 12, 13, and 24, respectively. tert-Butyldimethylsily-
lation of 25 and 26 afforded silyl ethers 29 and 30, which
underwent deacetonation, regioselective acetylation, oxidation,
and desilylation to give enone-alcohol 9 and 14, respectively.
Acidic removal of the trans-diacetal group in 9 gave 4-epi-
gabosine D (2).
RESULT AND DISCUSSION
■
Gabosines G (1)9a and D (3)9b were prepared as reported
previously (Figure 2). The syntheses of 4-epi-gabosine D (2)
To date, ethacrynic acid with a Michael acceptor, and some of
its analogues have been demonstrated to effectively inhibit various
GSTs.1b,11,7c There are two possible mechanisms for inhibition
of GST isozymes: formations of the enzyme−ethacrynic acid
adduct1b and GS−ethacrynic acid conjugate.1b,7c Because gabosine,
bearing the trihydroxycyclohexenone skeleton with a reactive
Michael acceptor, represents a promising core structure, we chose
it as the key compound for the development of GST inhibitors.
Figure 2. Structures of gabosine analogues 1−3.
and analogues 4−24 are briefly summarized in Scheme 1
(see Supporting Information for details). The known enone 4
was made from D-glucose via a key intramolecular direct aldol
Scheme 1. Syntheses of Gabosine Analogues
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In the initial screening of gabosine analogues, we identified that
gabosines 1−3 (Figure 2) could abolish GSTA2 and GSTP1-1
activities (Table 1, IC50 > 75 and >100 μM, respectively). To
enone 23 with a decyl ether group was the most active one
with respective IC50 values of 33.1, 33.6, and 36.5 μM toward
GSTA2, GSTM1, and GSTP1-1. Acid hydrolysis of diacetal-
enone 23 was achieved to give diol-enone 24, which showed
a potent inhibitory activity against GSTM1, with an IC50
value of 13.4 μM, and a similar inhibitory potency
toward GSTA2 and GSTP1-1 (IC50 = 37.8 and 25.3 μM,
respectively). Because the overexpression of different GSTs
in tumor cells is known, this has spawned the development
of new chemical entity to inhibit multiple GST isozymes.
4-O-Decyl-gabosine D (24) represents an example of a
promising new class of potent human GST inhibitors and is
used in this study.
For inhibition characteristics toward GSTM1, steady-state
kinetic analysis showed that 24 was competitive versus GSH
(Ki = 3.2 μM) but was noncompetitive versus CDNB (Ki =
4.7 μM; Table 2 and Figure 5). These results indicate that
inhibitor 24 competes with GSH for the hydrophilic binding
site (G-site), suggesting that gabosine D could be a new
alternative use for GSH in GST inhibitor design.
Maintaining a simple GST-mediated drug resistance rather
than a direct cytotoxic effect (from 24) is generally a
prerequisite for this study. To eliminate a direct cytotoxic
effect from GST inhibitor, the effect of 24 was tested on
proliferative inhibition against A549 (human lung adenocarci-
noma epithelial cell line) and OVCAR-3 (human ovarian
carcinoma cell line) cells to identify a suitable concentration for
this purpose. As shown in Figure 6, 24 exhibited negligible
effect on cell growth inhibition in the range 0−30 μM,
suggesting that exclusive GST isozymes inhibition could be
achieved with 30 μM 24.
As a first step toward understanding the effect for the reversal
of drug resistance by specifically inhibiting GST isozymes, we
evaluated the impact of 24 (0 and 30 μM) on anticancer drugs
(cisplatin, oxaliplatin, and doxorubicin) cytotoxicity against
A549, in vitro, using MTT assays over a period of 48 h
(Table 3). No effects were observed when 24 was incubated
with oxaliplatin or doxorubicin. However, a significant
enhancement of cisplatin-induced inhibition of cell viability
was detected at 30 μM 24, up to 270% (i.e., decrease in IC50
value by 2.7-fold) against A549 (Table 3). Surprisingly, no
effects were found in ovarian carcinoma when 24 was incubated
with anticancer drugs (Supporting Information Table S1).
Collectively, the results demonstrate that 24 specifically
enhances the antitumor efficacy of cisplatin in lung cancer
cells (not ovarian carcinoma) by blocking GST-mediated drug
resistance pathway rather than implicating a direct cytotoxic
effect.
Table 1. Inhibitory Constants of Gabosine Analogues 1−20
a
against different GST Isozymes
inhibitory activity, IC50 (μM)
compd
GSTA2
GSTM1
GSTP1−1
b
b
b
b
b
b
b
b
b
b
b
1
>100 (18)
>100 (27)
>100 (12)
>100 (16)
>100 (19)
>100 (17)
>100 (16)
>100 (20)
>100 (15)
>100 (16)
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
>100 (20)
b
2
>100 (25)
b
3
>100 (11)
b
4
>100 (15)
5
c
b
6
>100 (7)
b
7
>100 (23)
8
24.7 ± 2.1
b
9
>100 (16)
b
10
11
12
13
14
15
16
17
18
19
20
>100 (38)
24.5 ± 1.7
b
b
b
>100 (16)
>100 (14)
c
>100 (12)
16.3 ± 1.9
82.5 ± 5.3
62.7 ± 8.4
20.2 ± 0.8
48.2 ± 4.2
37.6 ± 6.3
52.4 ± 6.6
179.0 ± 29.1
c
b
>100 (10)
77.6 ± 4.4
96.3 ± 5.2
93.0 ± 2.9
178.6 ± 23.2
56.6 ± 9.2
162.7 ± 52.1
123.5 ± 16.1
176.7 ± 25.8
a
Inhibitor concentration at which half-maximal enzyme activity is
b
obtained (IC50, mean ± SEM, n = 3). The percent (%) inhibition, in
parentheses, at 100 μM is expressed as the percent (%) inhibition of
enzyme activity without correction for the contribution of the
c
formation of glutathione conjugate. Not determined.
enhance the potency of enones 1−3, we initially designed
and synthesized 13 compounds 4−16 with appropriately
protected hydroxy units and methyl/benzyl ether moieties
that had been selected to modulate hydrophobicity (Figure 3
and Scheme 1). This exercise to improve the IC50 was
successful. For example, alcohol 14 and methyl ether 15,
gabosine D (3) analogues, showed an acceptable inhibitory
activity against GSTP1-1 with IC50 values of 82.5 and
62.7 μM, respectively (Table 1). Similarly, benzyl-diol 8 and
benzyl-diacetal 11 (IC50 = 24.7 and 24.5 μM), 4-epi-gabosine
D (2) analogues, exhibited a 2−3-fold potency increase over
14 and 15. Furthermore, benzyl-diol 13 and benzyl-diacetal
16, the 4-epimers of 8 and 11, respectively, were found to
have IC50 values of 16.3 and 20.2 μM, representing 4−5-fold
higher potency than 14 and 15. In contrast, this series
displayed weak inhibitory activity against human GSTA2.
Collectively, the results show that the presence of a methoxy
or benzyloxy substituent in gabosine (especially for gabosine
D) at C-4 leads to significantly enhanced efficacy against
GSTP1-1, suggesting perhaps that a hydrophobic moiety at
position 4 could occupy a hydrophobic region in the active
site pocket of the enzyme. Building on these observations,
we therefore searched for hydrophobic moieties that can
improve structure activity profiles.
Interestingly, 24 has synergetic effect with cisplatin against
lung adenocarcinoma epithelial cell line through the inhibi-
tion of GSTs. Studies also showed different GST isozymes,
GSTA2, GSTM1, and GSTP1-1, to be overexpressed in A549
and OVCAR-3 (Supporting Information Table S2). This
raises the question of which GST isozymes are mainly
responsible for the resistance to cisplatin. To answer this
question, we depleted two GST mRNAs with small interfering
RNA (siRNAs) that were specific for GSTM1 and GSTP1-1
genes. To optimize the conditions of RNA interference
transfections, proteins levels were quantified by Western blot
analyses after treatment (Figure 7). By incorporating
si-GSTM1, the viability inhibition of cisplatin was enhanced
up to 138% (i.e., decrease in IC50 value by 1.4-fold) against
A549 (Table 3), suggesting that knockdown of GSTM1 gene is
Thus, seven analogues (17−23) related to benzyl-diacetal 16
(one of the most active compounds) with systematic variations
of the C-4 ether moiety were prepared (Figure 3, Scheme 1 and
Figure 4). Among these analogues (Table 1 and Table 2),
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Figure 3. Structures of gabosine analogues 4−20.
Figure 4. Rational design approach to develop a new class of GSTs inhibitors, gabosine D analogues.
responsible for major enhancement (∼50%) of 24-induced
synergetic effect with cisplatin. In contrast, transfection with
GSTP1 siRNA for 72 h had no effect on the viability of
A549 cells. These results suggest that other minor pathways
(e.g., inhibition of other GST isozymes, cellular stress, and
downregulation of MRP/P-gp) also play important roles in
sensitizing A549 cells to cisplatin-induced death. Neither
depletion of GSTP1 nor GSTM1 rendered cells more sensitive
to oxaliplatin and doxorubicin, which is apparently con-
sistent with the results shown by 24 (Table 3).
GSH and anticancer drugs. In particular, because the expression
of μ class of GSTs in COS and HeLa cells enhances cellular
resistance to cisplatin,12 lung, breast, and ovarian cancer
patients who are GSTM1-postive generally experience a
significant decrease in survival time and rate as compared to
the GSTM1-null patients.13 Our results are in line with the
above-mentioned conclusion that inhibition of GSTM1 is
essential for overcoming cisplatin resistance in lung cancer
A549 cells. However, overwhelming evidence (RNA interfer-
ence transfections, Table 3) suggests that other factors also play
important roles.
As with GSH, glutathione S-transferases contribute crucially
to drug resistance by catalyzing adduct formation between
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Table 2. Inhibitory Constants of Gabosine D (3) Analogues
21−24 against Different GST Isozymes
CONCLUSION
■
a
In summary, we have prepared and identified analogues of
natural product gabosine that serve as a new class of human
GST inhibitors. The findings highlight the utility of the
gabosine moiety for targeting the GSH binding site of GSTs. In
particular, the lead 24 shows synergetic effect with cisplatin
against lung cancer cell line, A549, through the inhibition of
GSTM1. These studies could potentially impact scientific areas
for potential development of bioactive compounds to overcome
GSTM1-related drug resistance and cellular stress signaling.
inhibitory activity, IC50 (μM)
compd
GSTA2
GSTM1
GSTP1-1
21
22
23
94.2 ± 4.8
66.3 ± 10.0
33.1 ± 1.4
44.5 ± 5.6
44.7 ± 4.9
33.6 ± 2.7
13.4 ± 2.5
(3.2 ± 0.8)
(4.7 ± 2.1)
58.0 ± 2.5
186.7 ± 18.2
36.5 ± 1.0
b
b
24
37.8 ± 2.0
25.3 ± 1.9
a
Inhibitor concentration at which half-maximal enzyme activity is
b
EXPERIMENTAL SECTION
obtained (IC50, mean ± SEM, n = 3). Values reported are Ki.
■
General Procedure. Melting points were measured with a
Reichert apparatus in Celsius degrees and are uncorrected. Optical
rotations were obtained with a Perkin-Elmer model 341 polarimeter,
operating at 589 nm. Infrared (IR) spectra were recorded on a Nicolet
205 or a Perkin-Elmer 1600 FT-IR spectrophotometer as thin films on
potassium bromide discs. Nuclear magnetic resonance (NMR) spectra
were measured with a Bruker DPX300 NMR spectrometer at
300.13 MHz (1H) or 400 MHz (1H) and at 75.47 MHz (13C)
or 100 MHz (13C) in CDCl3 solutions, unless stated otherwise. All
chemical shifts were recorded in ppm relative to tetramethylsilane (δ =
0.0). Spin−spin coupling constants (J value) recorded in Hz were
measured directly from the spectra. MS and HRMS were measured on
a ThermoFinnigan MAT 95 KL at the Department of Chemistry, The
Chinese University of Hong Kong. All reactions were monitored by
analytical thin-layer chromatography (TLC) on Merck aluminum-
precoated plates of silica gel 60 F254 with detection by spraying with
5% (w/v) dodecamolybdophosphoric acid in ethanol or 5% (w/v)
ninhydrin in ethanol and subsequent heating. E. Merck silica gel
60 (230−400 mesh) was used for flash chromatography. All reagents
and solvents were general reagent grade unless otherwise stated. THF
was freshly distilled from Na/benzophenone ketyl under nitrogen.
Dichloromethane was freshly distilled from P2O5 under nitrogen.
Other reagents were purchased from commercial suppliers and were
used without purification. Analytical HPLC was performed as follows:
eluent = MeOH: H2O (1:1); flow rate = 1.0 mL/min; Alltima C18 5 μ
250 mm × 4.6 mm column; detection at 254 nm. The purity of all the
Figure 5. Lineweaver−Burk inhibitory analysis of GSTM1 steady-state
kinetics by compound 24. (a) Reciprocal velocity versus reciprocal
GSH concentration with inhibitor 24 (4, 8, 12, 16, 20 μM). CDNB
concentration was held constant at 1 mM. (b) Reciprocal velocity
versus reciprocal CDNB concentration with inhibitor 24 (4, 6.5, 13, 26
μM). GSH concentration was held constant at 2 mM.
1
final gabosine analogues 1−24 was established as being >95% by H
NMR and HPLC analysis.
Synthesis of 28i (R = Decyl). Sodium hydride (60%, 69.2 mg,
1.73 mmol) was suspended in dry THF (5 mL) under nitrogen at
0 °C. A solution of the α-alcohol 26 (119.6 mg, 0.36 mmol) in THF
(5 mL) was added dropwise over 1 h at 0 °C, and then the mixture
was stirred for 1 h at 0 °C. 1-Bromodecane (0.23 mL, 1.11 mmol) was
added dropwise over 15 min, and tetra-n-butylammomium iodide
(2.67 mg, 0.067 mmol) was then added. The reaction mixture was
heated at reflux at for 1 h. After cooling to room temperature, water
was added slowly at 0 °C to destroy the excess of hydride, and this was
followed by the addition of saturated NH4Cl solution. The aqueous
Figure 6. In vitro cell viability effects of compound 24. Data are
representative of three individual experiments, performed in three
replicates (IC50, mean ± SEM, n = 3).
Table 3. Synergetic Effects of 4-O-Decyl-Gabosine D (24), GSTP1si and GSTM1si on Cell Viability of Human Lung Cancer
a
Cell Line A549
b
IC50 (μM)
drug
control
30 μM [24]
GSTP1si
GSTM1si
cisplatin
32.3 ± 3.9
59.5 ± 1.7
0.51 ± 0.06
12.0 ± 1.1
55.2 ± 5.9
0.64 ± 0.04
38.3 ± 1.0
56.4 ± 3.6
0.65 ± 0.12
23.4 ± 1.2
58.3 ± 3.4
0.52 ± 0.03
oxaliplatin
doxorubicin
a
A dose-dependent inhibitory effect on cell growth was observed and respective IC50 values of cisplatin, oxaliplatin, and doxorubicin were
determined after 48 h. For example, IC50 values of cisplatin were obtained in the absence (IC50 = 32.3 μM) or presence (IC50 = 12.0 μM) of 30 μM
4-O-decyl-gabosine D (24). This means that the presence of 24 reduces the dosage of cisplatin and enhances the efficacy of cisplatin to inhibit the
same amount of cancer cells growth. The calculation of 270% (2.7 × 100%) was derived from 2.7-fold increase of IC50 values from 32.3 μM to
b
12.0 μM (32.3/12.0 = 2.7). Cell proliferation was determined by measuring the absorbance 540 nm (MTT assay). Data are representative of three
individual experiments, performed in three replicates (IC50, mean ± SEM, n = 3).
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Figure 7. Conditions of depletion of GSTP1 and GSTM1 after treatment with siRNA for 48 and 72 h in A549 cells. Expression levels of GSTP1
(a) and GSTM1 (b) proteins were quantified by Western blot analysis.
layer was extracted with Et2O (3 × 100 mL). The combined organic
extracts were washed with brine, dried over MgSO4, and filtered. Con-
centration of the filtrate followed by flash chromatography (hexane:-
dichloromethane, 1:5) gave decyl ether 28i (127 mg, 75%) as a
colorless oil: [α]D20 −72.6 (c 1.98, CHCl3); Rf 0.4 (hexane:Et2O, 1:1).
NaHCO3 (10 mL). The organic layer was washed with brine (2 × 10
mL), dried (MgSO4), and filtered. Concentration of the filtrate
followed by flash chromatography (hexane:EtOAc, 2:1) gave acetate
34i (102.3 mg, 89%) as a colorless oil: [α]D20 −52.4 (c 0.57, CHCl3); Rf
0.52 (hexane:EtOAc, 1:1). IR (thin film) 3481, 2925, 1744, 1458,
1374, 1232, 1124, 885 cm−1. 1H NMR (300 MHz, CDCl3) δ 0.87 (3H,
t, J = 6.9 Hz), 1.26 (14H, m), 1.30 (3H, s), 1.32 (3H, s), 1.54−1.57
(2H, m), 2.06 (3H, s), 2.62 (1H, d, J = 4.8 Hz), 3.25 (3H, s), 3.29
(3H, s), 3.50−3.57 (1H, dt, J = 9, 6 Hz), 3.60 (1H, dd, J = 11.1, 3.9
Hz), 3.81−3.87 (1H, m), 3.91, (1H, dd, J = 5.4, 3.9 Hz), 4.09 (1H, dd,
J = 10.8, 8.1 Hz), 4.18−4.21 (1H, m), 4.50 (1H, d, J =
12.9 Hz), 4.88 (1H, d, J = 12.9 Hz), 5.87 (1H, d, J = 5.1 Hz). 13C
NMR (75.47 MHz, CDCl3) δ 14.5, 18.1, 18.2, 21.3, 23.1, 26.5, 29.8,
30.0, 30.1, 30.2, 30.7, 32.3, 48.3, 48.4, 64.4, 69.4, 70.2, 70.7, 72.4, 72.5,
99.2, 99.5, 125.5, 137.9, 171.4. MS (ESI) m/z (relative intensity) 495
([M + Na]+, 100). HRMS (ESI) calcd for C25H44O8 [M + Na]+
495.2928, found 495.2931.
1
IR (thin film) 2992, 2926, 1462, 1374, 1199, 1091, 852 cm−1. H
NMR (300 MHz, CDCl3) δ 0.86 (3H, t, J = 6 Hz), 1.25 (14H, m),
1.29 (3H, s), 1.30 (3H, s), 1.36 (3H, s), 1.48 (3H, s), 1.51−1.59 (2H,
m), 3.25 (3H, s), 3.26 (3H, s), 3.47−3.57 (1H, dt, J = 9, 6 Hz), 3.62
(1H, dd, J = 11.1, 3.9 Hz), 3.79−3.88 (2H, m), 4.07 (1H, d, J =
8.1 Hz), 4.13 (1H, d, J = 4.2 Hz), 4.34−4.35 (1H, m), 4.38 (1H, d, J =
9 Hz), 5.51 (1H, d, J = 4.8 Hz). 13C NMR (75.47 MHz, CDCl3) δ
14.5, 18.1, 18.2, 20.7, 23.1, 26.5, 28.4, 29.8, 30.0, 30.1, 30.7, 32.3, 48.3,
48.4, 63.3, 67.8, 69.5, 70.7, 72.3, 73.2, 98.9, 99.4, 99.5, 119.3 135.5. MS
(ESI) m/z (relative intensity) 493 ([M + Na]+, 100). HRMS (ESI)
calcd for C26H46O7 [M + Na]+ 493.3136, found 493.3119.
Synthesis of 23. A mixture of 3 Å molecular sieves (ca. 100 mg)
and pyridinium dichromate (PDC) (122.4 mg, 0.33 mmol) was added
to a solution of the acetate 34i (353.4 mg, 0.75 mmol) in dry CH2Cl2
(30 mL) under N2 at 0 °C. The mixture was stirred for 20 h at room
temperature. The mixture was then filtered through a pad of Celite.
and the residue was washed with EtOAc until no product was observed
in the eluent (checked with TLC). Concentration of the filtrate
followed by flash chromatography (hexane:Et2O, 2:1) yielded enone
23 (348.8 mg, 99%) as a colorless oil; [α]D20 −17.1 (c 2.49, CHCl3); Rf
0.37 (hexane:Et2O, 1:2). IR (thin film) 2926, 2855, 1748, 1461, 1376,
Synthesis of 33i. A solution of the alkyl ether 28i (189.9 mg,
0.40 mmol) in 80% aqueous AcOH (4.5 mL) was stirred at room
temperature for 1.5 h. The solvent was removed under reduced
pressure, and the residue was purified by flash chromatography
(hexane:EtOAc, 1:1) to afford diol 33i (150.5 mg, 87%) as a colorless
oil: [α]D20 −53.7 (c 1.29, CHCl3); Rf 0.47 (EtOAc). IR (thin film)
1
1139, 950 cm−1. H NMR (300 MHz, CDCl3) δ 0.86 (3H, t, J =
6.9 Hz), 1.25−1.31 (17H, m), 1.37 (3H, s), 1.53−1.64 (2H, m), 2.07
(3H, s), 3.23 (3H, s), 3.28 (3H, s), 3.58−3.65 (1H, dt, J = 9, 6.3 Hz),
3.92−4.01 (2H, m), 4.18 (1H, dd, J = 6, 3.3 Hz), 4.70 (1H, dd, J =
14.1, 1.2 Hz), 4.77−4.82 (2H, m), 6.85 (1H, d, J = 6.3 Hz). 13C NMR
(75.47 MHz, CDCl3) δ 14.5, 18.1, 21.3, 23.1, 26.5, 29.8, 29.9, 30.0,
30.1, 30.7, 32.3, 48.4, 48.7, 62.0, 69.9, 70.1, 72.2, 72.1, 99.7, 100.2,
135.6, 140.0, 170.8, 193.5. MS (ESI) m/z (relative intensity) 493
([M + Na]+, 100). HRMS (ESI) calcd for C25H42O8 [M + Na]+
493.2772, found 493.2780.
1
3419, 2925, 1650, 1463, 1375, 1123, 885 cm−1. H NMR (300 MHz,
CDCl3) δ 0.86 (3H, t, J = 6.6 Hz), 1.25−1.31 (20H, m), 1.44−1.63
(2H, m), 2.73 (1H, brs), 3.07 (1H, d, J = 5.4 Hz), 3.24 (3H, s), 3.28
(3H, s), 3.48−3.59 (2H, m), 3.80−3.94 (2H, m), 4.06 (1H, dd, J =
11.1, 8.1 Hz), 4.16−4.28 (3H, m), 5.79 (1H, d, J = 5.4 Hz). 13C NMR
(75.47 MHz, CDCl3) δ 14.5, 18.1, 18.2, 23.1, 26.5, 29.8, 30.0, 30.1,
30.7, 32.3, 48.2, 48.3, 64.1, 69.5, 70.4, 72.0, 72.4, 73.0, 99.1, 99.4,
122.6, 141.7. MS (ESI) m/z (relative intensity) 453 ([M + Na]+, 100).
HRMS (ESI) calcd for C23H42O7 [M + Na]+ 453.2823, found
453.2834.
Synthesis of 24. To a solution of the enone 23 (7.5 mg, 0.016
mmol) in CH2Cl2 (5 mL) were added trifluoroacetic acid (TFA) (0.1 mL)
and H2O (0.01 mL), and the mixture was stirred at room temperature
for 3 h. The solvent was removed under reduced pressure and flash
chromatography (hexane:EtOAc, 1:2) of the residue afforded diol 24
(5 mg, 88%) as a white solid: mp 47.8−48.2 °C; [α]D20 +114 (c 0.21,
CHCl3); Rf 0.5 (EtOAc). IR (thin film) 3417, 2924, 2854, 1747, 1692,
1
1369, 1228, 1102 cm−1. H NMR (300 MHz, CD3OD) δ 0.90 (3H, t,
J = 6.9 Hz), 1.29 (14H, m), 1.58−1.67 (2H, m), 2.07 (3H, s), 3.65
(1H, dt, J = 9.3, 6.6 Hz), 3.66 (1H, dt, J = 9.3, 6.6 Hz), 3.90 (1H, dd,
J = 9.3, 3.9 Hz), 4.26 (1H, dd, J = 4.8, 3.9 Hz), 4.31 (1H, d, J = 9.3 Hz),
4.74 (2H, s), 7.02−7.048 (1H, dt, J = 4.8, 1.5 Hz). 13C NMR (75.47
MHz, CD3OD) δ 14.4, 20.6, 23.7, 27.1, 30.4, 30.6, 30.7, 31.1, 33.0,
61.6, 72.3, 73.6, 75.1, 75.3, 135.4, 143.8, 172.1, 198.5. MS (ESI) m/z
(relative intensity) 379 ([M + Na]+, 100). HRMS (ESI) calcd for
C19H32O6 [M + Na]+ 379.2091, found 379.2098. HPLC analysis
indicated that it is free of any diastereomer and purity was determined
to be ≥95%.
Synthesis of 34i. To a solution of the diol 33i (121.7 mg,
0.28 mmol) and 2,4,6-collidine (0.11 mL, 0.83 mmol) in dry CH2Cl2
(10 mL) at −78 °C was added acetyl chloride (AcCl) (28 μL,
0.39 mmol) slowly. The reaction mixture was stirred for 15 h at −78 °C
and quenched with water (5 mL). The resultant solution was allowed to
warm to room temperature. The aqueous phase was extracted with EtOAc
(3 × 20 mL). The combined organic extracts were then washed with cold
1N HCl (2 × 10 mL), cold DI water (10 mL). and cold diluted
Cell Lines. Human lung cancer A549 cells (BCRC 60074) and
ovarian carcinoma OVCAR-3 cells (BCRC 60551) were purchased
from the Food Industry Research and Development Institute
(Hsinchu, Taiwan). The cells were grown in DMEM medium
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Journal of Medicinal Chemistry
Article
containing 10% (v/v) FBS, 1.5 g/L NaHCO3, 5 mM HEPES, 1 mM
sodium pyruvate, or RPMI1640 medium containing 20% (v/v) FBS,
1.5 g/L NaHCO3, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium
pyruvate, and supplemented with 0.01 mg/mL bovine insulin in a
humidified incubator under 5% CO2 and 95% air at 37 °C.
Gabosine Analogue Inhibitory Effect on Cell Viability. Cell
proliferation was determined using [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide] (MTT) assay. Briefly, the cells
were cultured in 96-well plates at 4 × 103 cells/well in DMEM or
RPMI1640 for 24 h. Then, the cells were treated with compound 24
(30 μM) for 2 h, followed by adding various concentrations of
anticancer drugs (cisplatin, oxaliplatin, and doxorubicin) for 48 h. After
incubation, the medium was removed and cells were washed with PBS,
followed by incubating with MTT solution for 4 h. The medium was
removed, and the formazan was solubilized in DMSO and measured
spectrophotometrically at 570 nm. The percentage of viable cells was
estimated by comparing with untreated control cells.
Transient Transfection of siRNA against GSTP1 and GSTM1. The
plasmid containing siRNA against GSTP1 and GSTM1 (GSTP1si
and GSTP1si; Stealth RNAi) and Lipofectamine RNAiMAX
transfection reagent were purchased from invitrogen (Australia).
GSTP1si and GSTM1si stock solutions (20 μM) were diluted with
DEPC water to form 10 μM solutions. Lipofectamine RNAiMAX
transfection reagent was mixed with 10 μM GSTP1si or GSTM1si
incubated for 20 min. The final concentration of siRNA was
10 nM. The cells were incubated with GSTP1si and GSTM1si for
48 and 72 h.
Western Blotting. Cancer cells were lysed in lysis buffer
(iNtRON BIOTECHNOLOGY), followed by centrifugation for
30 min at 4 °C. The protein (20 μg) from the supernatant were
separated on SDS-polyacrylamide gel electrophoresis and transferred
to PVDF membrane. After blocking with TBS buffer (20 mM Tris−
HCl, 150 mM NaCl, pH 7.4) containing 5% nonfat milk, the
membrane was incubated with GSTP1, GSTM1, and actin antibody
overnight and then incubated with horseradish peroxidase-conjugated
antirabbit or antimouse IgG for 1 h, followed by visualization using an
ECL chemiluminescent detection kit (Amersham Co, Bucks, UK).
Data Analysis. Data were expressed as mean ± standard deviation
(mean ± SD) and analyzed with t-test. If P was less than 0.05,
differences were considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
Cytotoxicity, experimental procedures, compound character-
ization, and NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*For T.K.M.S.: phone, +852-2609 6344; fax, +852 2603-5057;
E-mail, tonyshing@cuhk.edu.hk (synthesis). For W.S.L.: phone,
+886 (2) 27898662; fax, +886 (2) 27831237; E-mail,
wenshan@chem.sinica.edu.tw.
■
ACKNOWLEDGMENTS
■
We are grateful for funding from Academia Sinica and the
National Science Council. This work was also supported by a
Strategic Investment Scheme administered by the Center of
Novel Functional Molecules and by a direct grant from The
Chinese University of Hong Kong.
ABBREVIATIONS USED
■
hGSTs, human glutathione S-transferases; MRP, multidrug-
resistance-related proteins; P-gp, P-glycoprotein; PDC, pyr-
idinium dichromate; GSH, glutathione; A549, human lung
adenocarcinoma epithelial cell line; OVCAR-3, human ovarian
carcinoma cell line; siRNA, small interfering RNA
REFERENCES
■
(1) (a) Tew, K. D. Glutathione-Associated Enzymes in Anticancer
Drug Resistance. Cancer Res. 1994, 54, 4313−4320. (b) Ang, W. H.;
Khalaila, I.; Allardyce, C. S.; Juillerat-Jeanneret, L.; Dyson, P. J.
Rational Design of Platinum(IV) Compounds to Overcome
Glutathione-S-Transferase Mediated Drug Resistance. J. Am. Chem.
Soc. 2005, 127, 1382−1383.
(2) Broxterman, H. J.; Giaccone, G.; Lankelma, J. Multidrug
Resistance Proteins and Other Drug Transport-Related Resistance to
Natural Product Agents. Curr. Opin. Oncol. 1995, 7, 532−540.
(3) (a) Hayes, J. D.; Pulford, D. J. The Glutathione S-Transferase
Supergene Family: Regulation of GST and the Contribution of the
Isoenzymes to Cancer Chemoprotection and Drug Resistance. Crit.
Rev. Biochem. Mol. Biol. 1995, 30, 445−600. (b) Townsend, D. M.;
Tew, K. D. The role of Glutathione-S-Transferase in Anti-Cancer Drug
Resistance. Oncogene 2003, 22, 7369−7375.
Cloning, Expression, and Purification of Human GSTM1.
Human GSTM1 was cloned, expressed, and purified by using the
previously described procedures.7d The resulting hGSTM1 was added
20% glycerol before storage at −80 °C.
(4) Nebert, D. W.; Vasiliou, V. Analysis of the Glutathione S-
Transferase (GST) Gene Family. Hum. Genomics 2004, 1, 460−464.
(5) (a) McIlwain, C. C.; Townsend, D. M.; Tew, K. D. Glutathione
S-Transferase Polymorphisms: Cancer Incidence and Therapy.
Oncogene 2006, 25, 1639−1648. (b) Lo, H. W.; Ali-Osman, F.
Genetic Polymorphism and Function of Glutathione S-Transferases in
Tumor Drug Resistance. Curr. Opin. Pharmacol. 2007, 7, 367−374.
(6) (a) Wu, Z.; Minhas, G. S.; Wen, D.; Jiang, H.; Chen, K.; Zimniak,
P.; Zheng, J. Design, Synthesis, and Structure−Activity Relationships
of Haloenol Lactones: Site-Directed and Isozyme-Selective
Glutathione S-Transferase Inhibitors. J. Med. Chem. 2004, 47, 3282−
3294. (b) Ricci, G.; De Maria, F.; Antonini, G.; Turella, P.; Bullo, A.;
Stella, L.; Filomeni, G.; Federici, G.; Caccuri., A. M. 7-Nitro-2,1,3-
Benzoxadiazole Derivatives, a New Class of Suicide Inhibitors for
Glutathione S-Transferases. Mechanism of Action of Potential
Anticancer Drugs. J. Biol. Chem. 2005, 280, 26397−26405. (c) Ang,
W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C. J.;
Lo Bello, M.; Parker, M. W.; Dyson, P. J. Rational Design of an
Organometallic Glutathione Transferase Inhibitor. Angew. Chem., Int.
Ed. Engl. 2009, 48, 3854−3857.
Expression and Purification of Human GSTA2. Human
GSTA2 was cloned, expressed, and purified by using the previously
described procedures and stored in 16% glycerol at −80 °C.7c
Human GST Activity. Human GST activity was determined
spectrophotometrically at 340 nm and according to the procedure of
Habig et al..14
Inhibition of Human GST Isoenzymes-IC50 and Ki Determi-
nations. Human GST M1 (32 ng) was incubated with inhibitor
(ranging from 0 to 200 μM) in 100 mM potassium phosphate buffer
pH 6.5 at 37 °C. The reaction was initiated with GSH (final 2 mM)
and monitored at 340 nm in a period of 3 min. IC50 values were then
calculated by nonlinear regression analysis of inhibition data.
Lineweaver−Burk inhibitory analyses were performed in the same
condition as GST inhibition assays. GST M1 steady-state kinetics by
inhibitor 24 was assayed with various concentration of compound 24
at either constant CDNB (1 mM) or constant GSH (2 mM)
concentrations. Lineweaver−Burk plots were plotted with reciprocal
velocity against reciprocal GSH/CDNB concentration, and the
corresponding Ki values were calculated by fitting inhibition data
with competitive/noncompetitive inhibition equations.
(7) (a) Lyttle, M. H.; Hocker, M. D.; Hui, H. C.; Caldwell, C. G.;
Aaron, D. T.; Engqvist-Goldstein, A.; Flatgaard, J. E.; Bauer, K. E.
8580
dx.doi.org/10.1021/jm201131n | J. Med. Chem. 2011, 54, 8574−8581

Journal of Medicinal Chemistry
Article
Isozyme-Specific Glutathione-S-Transferase Inhibitors: Design and
Synthesis. J. Med. Chem. 1994, 37, 189−194. (b) Jao, S. C.; Chen, J.;
Yang, K.; Li, W. S. Design of Potent Inhibitors for Schistosoma Japonica
Glutathione S-Transferase. Bioorg. Med. Chem. 2006, 14, 304−318.
(c) Lo, W. J.; Chiou, Y. C.; Hsu, Y. T.; Lam, W. S.; Chang, M. Y.; Jao,
S. C.; Li, W. S. Enzymatic and Nonenzymatic Synthesis of Glutathione
Conjugates: Application to the Understanding of a Parasite’s Defense
System and Alternative to the Discovery of Potent Glutathione S-
Transferase Inhibitors. Bioconjugate Chem. 2007, 18, 109−120. (d) Li,
W. S.; Lam, W. S.; Liu, K. C.; Wang, C. H.; Chang, H. C.; Jen, Y. C.;
Hsu, Y. T.; Shivatare, S. S.; Jao, S. C. Overcoming the Drug Resistance
in Breast Cancer Cells by Rational Design of Efficient Glutathione
S-Transferase Inhibitors. Org. Lett. 2010, 12, 20−23.
(14) Habig, W. H.; Jakoby, W. B. Assays for Determination of
Glutathione S-transferase. Methods Enzymol. 1981, 77, 398−405.
(8) Tatsuta, K.; Tsuchiya, T.; Mikami, N.; Umezawa, S.; Umezawa,
H.; Naganawa, H. KD16-U1, a New Metabolite of Streptomyces:
Isolation and Structural Studies. J. Antibiot. 1974, 27, 579−586.
(9) (a) Shing, T. K. M.; Cheng, H. M. Short Syntheses of Gabosine I
and Gabosine G from delta-D-Gluconolactone. J. Org. Chem. 2007, 72,
6610−6613. (b) Shing, T. K. M.; Cheng, H. M. Facile Syntheses of
(+)-Gabosines A, D, and E. Org. Biomol. Chem. 2009, 7, 5098−5102.
(c) Shing, T. K. M.; Cheng, H. M.; Wong, W. F.; Kwong, C. S. K.; Li,
J.; Lau, C. B. S.; Leung, P. S.; Cheng, C. H. K. Enantiospecific
Synthesis of Pseudoacarviosin as a Potential Antidiabetic Agent. Org.
Lett. 2008, 10, 3145−3148. (d) Shing, T. K. M.; So, K. H.; Kwok, W. S.
Carbocyclization of Carbohydrates: Diastereoselective Synthesis of
(+)-Gabosine F, (−)-Gabosine O, and (+)-4-Epi-gabosine O. Org.
Lett. 2009, 11, 5070−5073.
(10) (a) Huntley, C. F. M.; Hamilton, D. S.; Creighton, D. J.;
Ganem, B. Reaction of COTC with Glutathione: Structure of the
Putative Glyoxalase I Inhibitor. Org. Lett. 2000, 2, 3143−3144.
(b) Kamiya, D.; Uchihata, Y.; Ichikawa, E.; Kato, K.; Umezawa, K.
Reversal of Anticancer Drug Resistance by COTC Based on
Intracellular Glutathione and Glyoxalase I. Bioorg. Med. Chem. Lett.
2005, 15, 1111−1114.
(11) Burg, D.; Riepsaame, J.; Pont, C.; Mulder, G.; van de Water, B.
Peptide-Bond Modified Glutathione Conjugate Analogs Modulate
GSTπ Function in GSH-Conjugation, Drug Sensitivity and JNK
Signaling. Biochem. Pharmacol. 2006, 71, 268−277.
(12) (a) Puchalski, R. B.; Fahl, W. E. Expression of Recombinant
Glutathione S-Transferase pi, Ya, or Yb1 Confers Resistance to
Alkylating Agents. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2443−2447.
(b) Hao, X. Y.; Widersten, M.; Ridderstrom, M.; Hellman, U.;
̈
Mannervik, B. Co-Variation of Glutathione Transferase Expression and
Cytostatic Drug Resistance in HeLa Cells: Establishment of Class Mu
Glutathione Transferase M3-3 as the Dominating Isoenzyme. Biochem.
J. 1994, 297, 59−67.
(13) (a) Ge, H.; Lam, W. K.; Lee, J.; Wong, M. P.; Yew, W. W.;
Lung, M. L. Analysis of L-Myc and GSTM1 Genotypes in Chinese
Non-Small Cell Lung Carcinoma Patients. Lung Cancer 1996, 15,
355−366. (b) Nedelcheva, K. V.; Andersen, T. I.; Erikstein, B.;
Geitvik, G.; Skovlund, E.; Nesland, J. M.; Børresen-Dale, A. L. Single
Tube Multiplex Polymerase Chain Reaction Genotype Analysis of
GSTM1, GSTT1 and GSTP1: Relation of Genotypes to TP53 Tumor
Status and Clinicopathological Variables in Breast Cancer Patients.
Pharmacogenetics 1998, 8, 441−447. (c) Yang, P.; Yokomizo, A.;
Tazelaar, H. D.; Marks, R. S.; Lesnick, T. G.; Miller, D. L.; Sloan, J. A.;
Edell, E. S.; Meyer, R. L.; Jett, J.; Liu, W. Genetic Determinants of
Lung Cancer Short-Term Survival: The Role of Glutathione-Related
Genes. Lung Cancer 2002, 35, 221−229. (d) Matsumoto, T.; Hayase,
R.; Kodama, J.; Kamimura, S.; Yoshinouchi, M.; Kudo, T.
Immunohistochemical Analysis of Glutathione S-Transferase in
Expression in Ovarian Tumors. Eur. J. Obstet. Gynecol. Reprod. Biol.
1997, 73, 171−176. (e) Medeiros, R.; Pereira, D.; Afonso, N.;
Palmeira, C.; Faleiro, C.; Afonso-Lopes, C.; Freitas-Silva, M.;
Vasconcelos, A.; Costa, S.; Osório, T.; Lopes, C. Platinum/
Paclitaxel-Based Chemotherapy in Advanced Ovarian Carcinoma:
Glutathione S-Transferase Genetic Polymorphisms as Predictive
Biomarkers of Disease Outcome. Int. J. Clin. Oncol. 2003, 8, 156−161.
8581
dx.doi.org/10.1021/jm201131n | J. Med. Chem. 2011, 54, 8574−8581