Synthesis and antimultidrug resistance evaluation of icariin and its derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.05.103

A series of icariin derivatives were synthesized. Their multidrug resistance (MDR) reversal activities were evaluated by MTT assay and the results indicated that the derivatives were the potent modulators of MDR. It was showed that the derivatives signifi

Full text of DOI:10.1016/j.bmcl.2009.05.103

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4237–4240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimultidrug resistance evaluation of icariin and its derivatives
*
*
Dong-Fang Liu, Yan-Ping Li, Tian-Miao Ou, Shi-Liang Huang, Lian-Quan Gu , Min Huang, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of icariin derivatives were synthesized. Their multidrug resistance (MDR) reversal activities were
evaluated by MTT assay and the results indicated that the derivatives were the potent modulators of
MDR. It was showed that the derivatives significantly increased the intracellular accumulation of ADR
in MCF-7/ADR cells compared with drug sensitive MCF-7 cells. The results of bi-directional assay and
reverse transcription polymerase chain reaction (RT-PCR) assay showed that the derivatives had high
inhibitory activity against P-gp efflux function and significantly down-regulated on the expression of
P-gp.
Received 18 March 2009
Revised 20 May 2009
Accepted 25 May 2009
Available online 30 May 2009
Keywords:
Icariin derivatives
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
Multidrug resistance reversal activity
P-Glycoprotein inhibitors
Multidrug resistance (MDR) is a protecting action of a cancer
cell against a variety of drugs with different structures and func-
tions.1 MDR of cancer cells is thought of a major cause of failure
in chemotherapy. P-Glycoprotein (P-gp), a plasma membrane
transporter which extrudes chemotherapeutic agents out of cells,
this function is one of the mechanisms of MDR of cancer cell, and
the overexpression of this protein could be found in many cancer
cells with MDR.2 The mechanism of P-gp is cell- or tissue-specific,
such as the protection against toxic xenobiotics by blocking
absorption by the intestine, excretion of chemicals into the bile
duct or kidney tubules, prevention of chemicals taken into the
brain through the blood–brain barrier, and efflux of steroid hor-
mones and cholesterol from feces.3–5 Combined therapy with
MDR-related drugs and modulators is a promising strategy to over-
come clinical MDR.
A large number of compounds have been found to function as
inhibitors of P-gp.6,7 A few of these compounds are currently under
clinical evaluation. The first generation of MDR reversal agents such
as verapamil (VRP) and cyclosporine A, modulated MDR at very high
concentrations, resulting of enhanced cytotoxicity observed in nor-
mal cells. The second generation of MDR reversal agents such as dex-
niguldipine8 and dexverapamil9 were found to be more selective
against P-gp, while they still were not able to exhibit improved
potency. The acridone–carboxamide derivatives GG91810 and
PSC83311 displayed an activity that was 10–30 times more potent
than cyclosporin A.12 Problems of MDR reversal agents were higher
toxicity, lower inhibitory activity and with some side effects.
It has been identified that flavonoids possessed the ability to
bind with P-gp.13 Flavonoids are abundant in natural products
and believed to have medication potential. Epimedii is a traditional
Chinese medicine, which is widely used as a tonic aphrodisiac and
antirheumatic. A flavonoid icariin (compound 1), isolated from this
herb, had been proven to be effective against cardiovascular
disease and immune-regulation.
In order to synthesize lower toxic and higher potential inhibi-
tors of P-gp, the structural modifications of icariin were carried
out by two strategies. One was removing sugar groups that conju-
gated on icariin, since a P-gp inhibitory activity of aglycone 3 was
reported.13 The other was introducing sulfonyl groups in the
aglycones 3 and 5, which was expected to increase interactions
with P-gp in plasma membrane. Nine derivatives of icariin were
designed and synthesized using natural product icariin as starting
materials, as shown in Schemes 1 and 2, compound 2, 3, 4, 5 and 6
were obtained from icariin by hydrolysis in different conditions,
compound 7 and 8 were obtained by reaction of compound 3
and 5 with ClSO2CH3, respectively, compound 9 were synthesized
by reaction of 5 with ClSO2NH2. Derivative 10 was synthesized
from 5 to obtain better aqueous solubility. The antimultidrug resis-
tance activities of all compounds (1–10) were evaluated.
The MDR modulating activity of the derivatives was evaluated
by MTT assay using breast carcinoma cells MCF-7 and adriamycin
(ADR) resistant breast carcinoma cells MCF-7/ADR.14 The cultured
cell lines were treated with ADR in the presence or absence of the
derivatives, the concentration (
cell growth after 72 h of drug exposure were calculated as IC50
The results were summarized in Table 1. The IC50 for ADR to the
sensitive MCF-7 cells was 0.53 M, while to MCF-7/ADR cells was
25.28 M, the MDR of MCF-7/ADR cells was 47.7-fold higher com-
pared with the sensitive MCF-7 cells. As shown Table 1, icariin and
lM) required for 50% inhibition of
.
* Corresponding authors. Tel./fax: +86 20 39943056 (Z.-S. Huang); tel.: +86 20
39943055; fax.: +86 20 39943056 (L.-Q. Gu).
E-mail addresses: cesglq@mail.sysu.edu.cn (L.-Q. Gu), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
l
l
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.103

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D.-F. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4237–4240
OMe
OMe
OMe
OMe
Pr
Pr
Pr
Pr
HO
O
HO
O
Glu-O
O
Glu-O
O
a
b
+
OH
O-Rha
O-Rha
OH
OH O
3
OH O
OH O
OH O
2
4
1
c
e
d
OMe
OMe
OMe
Pm
Pr
HO
O
MeO2SO
O
O
O
Pr =
OH
OSO2Me
OH
OH O
OH O
7
OH O
Pm =
OMe
5
6
Scheme 1. Synthesis of icariin derivatives 2–7. Reagents and conditions: (a) b-glucosidase, pH 5.0 acetic acid buffer, 37 °C, 36 h; (b) H2SO4 (2–5 equiv), 90% EtOH, reflux;
1.5 h; (c) 20% H2SO4, MeOH, reflux, 2 h; (d) 20% H2SO4, MeOH, 25 °C, 4 h; (e) ClSO2CH3, Et3N, 4 h, then HCl.
OMe
OMe
OMe
O
O
O
O
O
O
O
g
f
OSO2Me
OSO2NH2
OH
OH
OH O
OH O
OH O
8
5
9
h
SO3H
OMe
O
OH O
10
Scheme 2. Synthesis of icariin derivatives 8–10. Reagents and conditions: (f) ClSO2CH3, pyridine, 0 °C, 3 h; then HCl; (g) ClSO2NH2, DMA, CH2Cl2, 0 °C, 3 h; (h) ClSO3H, Et3N,
0 °C, 3 h; then HCl.
its derivatives showed low cytotoxic activities against MCF-7/ADR
cells except compound 6, although they enhanced the cytotoxicity
of ADR in MCF-7/ADR cells to a certain level and reversed the MDR
in a concentration-dependent manner. Compound 3 and 5 showed
significant reversal activity, by increasing the sensitivity of the
MCF-7/ADR cells to ADR by about 10-fold. This suggested icariin
derivatives might be the potent P-gp inhibitors. In a structure–
activity relationship study, the effect of removing sugar suggested
a crucial role of hydrophobicity in P-gp modulation. Slightly effects
were observed upon introduction of sulfonyl groups, suggesting
that the free hydroxyl group of 3-position might be important.
Comparing analysis of the activity data and structures of com-
pounds 3, 5, and 6. We speculated that isoprenyl and cycloether
groups at 8-position might also be a key elements for the reversal
activity of derivatives.
To elucidate the mechanism of the MDR reversal activity of the
derivatives, the intracellular ADR accumulation was examined as
described by Fu et al.15 As shown in Figure 1, the intracellular accu-
mulation of ADR in MCF-7/ADR cells was only about 50% of that in
MCF-7 cells. Treatment with the derivatives increased the intracel-
lular accumulation of ADR at certain level. However, little effect
was observed in presence of the derivatives in drug sensitive
MCF-7 cells. It has been generally accepted that P-gp function as
an energy-dependent efflux pump by expelling cytoxic substances
and thereby decreasing drug accumulation of tumor cells. ADR was
known to be a substrate of P-gp. We speculated that icariin deriv-
atives modulated intracellular ADR levels by inhibiting the efflux
function of P-gp, thereby increasing the ADR accumulation in
MCF-7/ADR cells. Based on the results shown in Figure 1, com-
pounds 5, 8, 9, and 10, in which their 8-isoprenyl group formed a
cycloether, had higher activity in increasing the ADR accumulation
than other derivatives.
Bi-directional assay in Caco-2 cells was also performed to fur-
ther confirm the inhibition of the derivatives on P-gp efflux
function.16 A well characterized and well studied standard P-gp
substrate taxol was selected to run the bi-directional assay.
Compounds 3 and 5, which had more potential of P-gp inhibi-
tion, were applied to the assay. Ketoconazole (K), an inhibitor
of P-gp, was used as positive control of the experiment.17 As
shown in Figure 2, in the presence of this inhibitor, the influx
permeability (A to B) increased significantly along with
a
decrease in the efflux permeability (B to A). In the presence of
compound 3 and 5, taxol (T) demonstrated similar permeability
in both direction with K, the P-gp function was inhibited by
45.0% (ketoconazole), 41.9% (3), 39.6% (5). These results sug-
gested that the function of P-gp as efflux pump was inhibited
by synthesized icariin derivatives.
To investigate whether the derivatives also function in the
repression of MDR1 gene, which was responsible for the expres-
sion of P-gp, RT-PCR assay was performed.18,19 Five more active
derivatives (3, 5, 8, 9, and 10) in increasing the ADR accumulation
were chosen for this RT-PCR assay. The results in Figures 3 and 4
showed that the expression of MDR1 was obviously inhibited by
the testing derivatives.

D.-F. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4237–4240
4239
Table 1
Effects of compounds on the cytotoxicity of adriamycin to MCF-7/ADR cells
(A to B)
(B to A)
150
120
90
60
30
0
Modulators
Control
Concn
0
IC50
IC50
MFc
a
b
25.28
Verapamil
1
10
25
>100
>100
>100
52.09
60.78
>100
20.93
80.78
54.95
92.13
>100
8.97
3.75
2.36
2.82
6.74
10.71
1
2
3
4
5
6
7
8
9
10
1
10
25
17.88
15.87
12.45
1.41
1.59
2.03
1
10
25
19.76
18.06
15.75
1.28
1.39
1.61
1
10
25
10.19
5.10
1.96
2.48
4.96
12.89
T
T+k
T+3
T+5
1
10
25
17.97
16.98
15.59
1.41
1.49
1.62
Figure 2. Effect of compounds 3 and 5 on bi-directional permeability of taxol (T) in
the 12 well Caco-2 cell transwells. The bi-directional permeability of taxol was
assessed in the presence and absence of test compound, Taxol concentration was
10 lM and the test compounds were 50 lM in both compartments. Permeability
studies were performed at 37 °C for 2 h. Each column represents the mean of three
1
10
25
10.87
5.81
2.51
2.33
4.35
10.07
data points.
1
10
25
20.03
10.50
n.d.
1.26
2.41
n.d.
1
10
25
22.82
18.62
17.27
1.11
1.36
1.46
1
10
25
13.38
10.80
9.34
1.89
2.34
2.71
1
10
25
17.78
15.14
6.92
1.42
1.69
3.65
1
10
25
18.99
11.78
9.87
1.33
2.15
2.56
Figure 3. The transcription of MDR1 in the MCF-7/ADR cells were carried out in the
presence the derivatives at the 25
treatment of the derivatives.
lmol/l, The control was the cells without the
All values are mean of three independent experiments.
n.d. means ‘not determined’.
a
IC50 was intrinsic cytotoxicity of verapamil and the derivatives on MCF-7/ADR
cells.
b
IC50 was cytotoxicity of adriamycin on MCF-7/ADR cells in presence of the
derivatives.
MF was expressed as the modulation factor, defined as the ratio of the IC50 for
ADR alone versus the IC50 for ADR in the presence of the modulators.
100
80
60
40
20
0
c
MCF-7/ADR
MCF-7
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
control
3
5
8
9
10
Figure 4. The signal of MDR1 and b-actin was quantified with Alphaimager
software (Alpha INNOTECH) and the background was subtracted. The MDR1 signal
was normalized to actin and the value for control was set as 100%.
In conclusion, semi-synthesized icariin derivatives displayed a
potential MDR reversal activity, and the inhibitory ability should
be attributed to the inhibition of the P-gp efflux function and down
regulation of P-gp gene expression. These results indicated that
these compounds are potential MDR reversal agents and are prom-
ising for further development.
cont.VRP
1
2
3
4
5
6
7
8
9
10 --
Figure 1. Effect of icariin and its derivatives (10
lM) on intracelluar ADR
accumulation in MCF-7 cells and MCF-7/ADR cells.

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