Design, synthesis and evaluation of a novel series of inhibitors reversing P-glycoprotein-mediated multidrug resistance

Article abstract of DOI:10.1111/cbdd.13338

Multidrug resistance (MDR) is still the main barrier to attaining effective results with chemotherapy. Discovery of new chemo-reversal agents is needed to overcome MDR. Our study focused on a better way to obtain novel drugs with triazole rings that have an MDR reversal ability through click chemistry. Among 20 developed compounds, compound 19 had a minimal cytotoxic effect compared to tariquidar and verapamil (VRP) and showed a higher reversal activity than VRP through increased accumulation in K562/A02 cells. Compound 19 also played an important role in the P-gp efflux function of intracellular Rh123 and doxorubicin (DOX) accumulation in K562/A02 cells. Moreover, compound 19 exhibited a long lifetime of approximately 24?hr. These results indicated that compound 19 is a potential lead compound for the design of new drugs to overcome cancer MDR.

Full text of DOI:10.1111/cbdd.13338

PROFESSOR HAI QIAN (Orcid ID : 0000-0002-3827-0992)
Article type : Research Article
Design, Synthesis and Evaluation of a Novel Series of Inhibitors
Reversing P-Glycoprotein-Mediated Multidrug Resistance
a
a
a, 1
a
a
Hesham Ghaleb , Huilan Li a, 1, Mutta Kairuki , Qianqian Qiu , Xinzhou Bi , Chunxia Liu , Chen
a
a, c*
a
b
,
a, c*
Liao , Jieming Li , Kamal Hezam , Wenlong Huang
Hai Qian
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Antibody Engineering Laboratory, School of Life Science and Technology, China
Pharmaceutical University, Nanjing 210009, China
c Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
Abstract
Multidrug resistance (MDR) is still the main barrier to attaining effective results with
chemotherapy. Discovery of new chemo-reversal agents is needed to overcome MDR. Our
study focused on a better way to obtain novel drugs with triazole rings that have an MDR-
*
Corresponding authors at: Centre of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com (H. Qian).
1 These authors contributed equally to this work
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13338
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reversal ability through click chemistry. Among 20 developed compounds, compound 19 had
a minimal cytotoxic effect compared to tariquidar and verapamil (VRP) and showed a higher
reversal activity than VRP through increased accumulation in K562/A02 cells. Compound 19
also played an important role in the P-gp efflux function of intracellular Rh123 and
doxorubicin (DOX) accumulation in K562/A02 cells. Moreover, compound 19 exhibited a
long lifetime of approximately 24 h. These results indicated that compound 19 is a potential
lead compound for the design of new drugs to overcome cancer MDR.
Keywords: MDR; P-gp inhibitor; Click chemistry; Reversal activity.
Introduction
Cancer has devastated the lives of many people worldwide, and multidrug resistance
(MDR) is mainly accountable for the failure of chemotherapy in over 90% of metastatic
cancer patients. Whether acquired during treatment or inherent with the disease being
refractory to chemotherapy from the outset, [1-4] MDR is caused by reduced apoptosis, altered
drug metabolism or DNA damage repair mechanisms and the active efflux of drugs by ATP-
binding cassette (ABC) transporters. Overexpression of P-glycoprotein (P-gp) in cancer cells
is the main aetiological reason for the enhancement of MDR.[5, 6]
P-glycoprotein (P-gp) is the primary human ABC drug transporter. It utilizes ATP hydrolysis
[7, 8]
for energy to pump drugs out of cells and has diverse substrate specificities.
Co-
administration of a P-gp inhibitor together with the primary chemotherapeutic agent is
recommended as an effective strategy to overcome MDR. [9] Currently, three generations of
P-gp inhibitors have been developed, with the first generation being VRP and cyclosporine,
[10, 11]
the second generation consisting of dexverapamil, valspodar (PSC 833),
and the third
generation being tariquidar[12] and WK-X-34[13] (Figure 1). However, a series of side effects
due to high toxicity, low specificity and drug interactions have limited the development of
first- and second-generation inhibitors. Third-generation MDR inhibitors have attracted
attention due to their high potency and specificity.[4] To date, no P-gp inhibitors have been
approved for therapeutic application.[14] Therefore, it is still imperative to develop novel P-gp
inhibitors with better safety and higher activities for use against cancer MDR.
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In this paper, we sought to develop potent P-gp inhibitors though structure modification by
the introduction of a triazole ring to a potent third-generation P-gp inhibitor, tariquidar. In the
experiments 1H-1,2,3-triazole rings were introduced as a bioisostere of the carboxamide
group in the designed compounds through click chemistry methods commonly applied in
drug discovery and amide reversal.[15, 16] Finally, we obtained a novel series of P-gp inhibitors
with a triazol-phenethyl-tetrahydroisoquinoline scaffold (Figure 2). All of the target
compounds were evaluated for cytotoxicity and the ability to reverse P-gp-mediated MDR
against doxorubicin (DOX). Among the active compounds, compound 19 had low cytotoxic
activity and potent reversal activity on resistant cancer cells, and this compound was chosen
for further evaluation at different concentrations. Additionally, the in-depth studies
demonstrated that compound 19 had an extraordinary inhibition effect on P-gp transport
activity and persistent duration of its reversal effect, which indicates promising potential.
2. Chemistry
The synthetic routes to target compounds 1-20 are shown in Scheme 1. First, methyl 2-
amino-4,5-dimethoxybenzoate and 3-bromo-prop-1-yne were refluxed in a mixture of DMF
and potassium carbonate for 6 h to afford compound a. Second, compound a was reacted
with lithium hydroxide in 75% methanol to get compound b, which was then treated with the
substituted amine (DIEA) in dry dichloromethane (CH2Cl2), followed by EDCI and HOBT to
give compounds c1-c20. Compounds d-f were synthesized according to literature procedures
with slight modifications. [16, 17] Compounds c1-c20 and compound f were treated with sodium
ascorbate and copper sulfate in 75% methanol with stirring at room temperature for 24-48 h
to provide compounds 1-20. All compounds were purified by column chromatography. The
structures of the target compounds obtained are listed in Scheme 1.
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3. Results and discussion
3.1. Biological evaluation
3.1.1. Determination of the Intrinsic Cytotoxicity via MTT Assay.
A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used
to evaluate the cytotoxicity of the newly synthesized compounds against human
erythroleukaemia K562 cells and DOX-resistant K562/A02 cells. The chemotherapy drugs
DOX, VRP and tariquidar were selected as controls. As shown in Table 1, VRP possessed
weak cytotoxic effects towards K562 and K562/A02 cells with IC50 values of 70.13 and 61.51
µM, respectively. Tariquidar showed a high level of toxicity towards K562 cells (IC50 of
30.77 µM) and K562/A02 cells (IC50 of 28.8 µM). In contrast, all of the synthesized
compounds displayed no toxicity, with IC50 values higher than 100 µM for both types of cells
(K562/A02 and K562).
3.1.2 Effects of the target compounds on reversal of DOX resistance in K562/A02 cells.
Based on the cytotoxicity testing experiment, all of the target compounds were found to have
low cytotoxicity in the tested cell lines. To evaluate the activity of the target compounds, we
tested their effects on reversal of DOX resistance in P-gp-overexpressing drug-resistant
[18, 19]
leukaemic K562/A02 cells by the MTT method.
We selected verapamil (VRP) and
tariquidar as positive controls. Compounds 1–20 and the positive controls were tested at 4
μM. The IC50 values of DOX combined with individual compounds against each tumour cell
line are depicted in Table 2. The anticancer drug DOX alone demonstrated a slight effect on
K562/A02 cells (half-maximal inhibitory concentration IC50 = 35.9±2.1 μM). However, co-
administration of DOX and target compounds, VRP or tariquidar led to an increase of the
inhibitory effects on K562/A02 cells to different extents, suggesting that all of the test
compounds could reverse DOX resistance. Compound 19 showed the most potent reversal
fold (RF = 31.2), which was higher than that of VRP (RF = 8.2). Therefore, compound 19
may represent a promising candidate for developing a P-gp-mediated MDR inhibitor.
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3.2. Structure–activity relationships
Analysis of the structure-activity relationships (SARs) was based on the results in Table 2.
Different substituents in the R groups of compounds 1-20 could affect the MDR reversal
activities in K562/A02 cells. Compound 1 showed the least activity among the twenty
compounds, and it can be concluded that the absence of a benzene ring led to a decrease in
activity. Meanwhile, the presence of a benzene ring produced increases in efficiencies, such
as that seen with compound 2. It was also observed that compounds bearing electron-
donating groups in R showed better MDR reversal activities than those with electron-
withdrawing groups. For example, compounds 3, 4, 5 and 6, which possessed electron-
donating groups like -CH2CH3, -CH (CH3)2, -CH3 and -C (CH3)3, showed higher MDR
reversal activities. Additionally, the size of the substituent in the R group is likely to affect
the MDR reversal activity. For example, compound 6, containing a 4-tert-butyl group in R,
was more potent than compounds 3, 4 and 5, which had other alkyl substituents. Interestingly,
in spite of having the same molecular weight (704, 8) and the same alkyl substituent position,
the activity of compounds 6 and 7 showed a slight difference produced from the arrangement
of the carbon chain in which the 4-tertiary butyl group of compound 6 was more active than
the n-butyl group of compound 7.
The substitution position of the electron-donating group in R also influences the reversal
activity, as in the effect seen with compounds 4 and 5, which showed that substitution in the
ortho position produced a more potent reversal effect than that of VRP. Moreover,
compounds 8, 9 and 10, which were substituted by -OCH3 in ortho, meta, and para positions
showed lower MDR reversal activities than VRP. In addition, compound 11, containing a
4,5-dimethoxy group in R, was more potent than compound 12, which contained a 3,4,5-
trimethoxy group. This difference may be related to the increased molecular weight.
Compounds 13, 14, 15 and 16 with electron-withdrawing groups displayed less MDR
reversal activity, and compound 17, which was substituted by -OCF3, presented poor MDR
reversal activity.
The absence of substituent groups on the benzene ring produced increases in efficiencies such
as those seen with compounds 18 and 19 (IC50s = 1.76 and 1.51 µM), in which compound 19
showed the best activity. Structurally, compound 19 has a -CH2CH2 linker between the
carbonyl group and R, but surprisingly, compound 18 with a single -CH2 linker and
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compound 20 with two methoxy groups on the benzene ring resulted in lower IC50 values of
2.34 and 1.92 µM, respectively, compared with that of compound 19.
To investigate and further characterize the interaction between the target compound and P-gp,
compound 19 was selected for a docking study with a model of P-gp (PDB code: 3G60). As
shown in Figure 3, the interaction mode of compound 19 occurred in a small hydrophobic
pocket produced by Tyr-949 and Phe-339.
3.1.3. Chemo-sensitizing effect of target compounds
Based on the above results, we chose the most potent compound 19 for further investigation
of reversal potency and dose–response effects. We examined the reversal activity of
compound 19 at numerous other concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.156 and
0.078 μM) in K562/A02 cells by an MTT assay, for which we selected VRP as a positive
[13]
control.
The results are summarized in Table 3. VRP displayed a slight modulating
activity at 5.0 µM (RF = 14.7). However, compound 19 showed apparent dose-dependent
activity and still displayed potent MDR reversal activity (RF = 38.3) when the concentration
was decreased to 2.5 µM. Additionally, the EC50 value of compound 19 was 306±8.6 nM,
which was calculated by GraphPad Prism 6.0 software from the dose-response curve as
shown in Figure 4. The results suggested that compound 19 possessed significant potential
to improve the sensitivity of P-gp-overexpression cells to anticancer drug substrates in a
dose-dependent manner.
3.1.4. Duration of the MDR reversal effect of compound 19 for DOX in K562/A02 cells
It is obvious that an ideal P-gp inhibitor should have a relatively long duration of reversal
[20]
action for safe and effective therapy of P-gp-mediated cancer MDR.
The assay was
performed as reported previously. [11] We selected VRP as a positive control to determine the
duration of the MDR reversal effect of compound 19. The duration time results are displayed
in Table 4. The reversal activity of 5.0 μM VRP totally disappeared after its removal from
the medium, indicating that the action of VRP appeared to persist no more than 6 h. In
contrast, a 5.0 μM concentration of compound 19 exhibited reversal activity even after its
removal from the medium for 24 h, and the IC50 of DOX was 33.67 µM (RF = 1.78). These
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data suggested that compound 19 revealed potent MDR-reversing effects that could persist
for a longer time compared with those of the positive control, VRP.
3.1.5. Effect of target compound 19 on DOX accumulation
To confirm our assumption, we assessed the accumulation of DOX, a fluorescent substrate of
P-gp, in K562/A02 cells by using laser confocal fluorescence microscopy. The classical P-gp
inhibitor VRP was selected as a positive control, as shown in Figure 5. Sensitive K562 cells
maintained most of the red fluorescence of DOX after incubation. In contrast, there was no
fluorescence of DOX observed in P-gp overexpressing K562/A02 cells in the absence of P-gp
inhibitors. This effect may have been due to the ability of P-gp to pump DOX out of the cells.
When K562/A02 cells were treated with compound 19 and VRP, the accumulation of DOX
was significantly increased in a dose-dependent manner, and the fluorescence intensity of
K562/A02 cells treated with compound 19 was higher than that of cells treated with VRP at
the same conditions. Therefore, the results indicated that compound 19 was more potent than
VRP in inhibiting the drug efflux function of P-gp.
3.1.6. Inhibitory effect of compound 19 on the P-gp efflux function
Furthermore, to confirm whether compound 19 could reverse the P-gp-mediated drug efflux
function, we selected rhodamine-123 (Rh123) as another fluorescent substrate of P-gp, which
was photographed via laser confocal fluorescence microscopy. As demonstrated in Figure 6,
the classical P-gp inhibitor VRP was chosen as a positive control. The sensitive K562 cells
maintained most of the fluorescence of Rh123, while the K562/A02 cells (P-gp
overexpression) did not show fluorescence of Rh123. In comparison, the fluorescence
intensity of Rh123 in K562/A02 cells was obviously increased in a dose-dependent manner
after treatment with compound 19 or VRP, but cells treated with compound 19 exhibited a
much higher level of Rh123 fluorescence than those treated with VRP at the same doses.
These results supported our presumption that compound 19 exhibited a stronger inhibitory
effect on the P-gp-mediated drug efflux function than the classical P-gp inhibitor VRP under
the same conditions.
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4. Conclusions
In summary, we selected tariquidar as a lead compound, from which twenty compounds were
newly designed and synthesized based on click chemistry. Our studies permitted us to
identify a new compound as a highly potent P-gp modulator in cancer cells. Based on the
results obtained, compound 19 exhibited a higher reversal activity than VRP through its
increased accumulation in K562/A02 cells and by blocking its efflux from the cells,
producing a much longer chemosensitizing effect (>24 h) than the known P-gp inhibitor VRP
(<6 h) with reversibility. Moreover, the cytotoxic effects of compound 19 were negligible
against all tested cell lines compared with those of tariquidar and VRP. Therefore, compound
19 can be considered promising for the development of P-gp-mediated MDR reversal agents.
5. Experimental section
5.1. Chemistry
1
The structures of the target compounds were characterized by H NMR and 13C NMR
spectroscopy (Bruker ACF-300Q, 300 MHz), with Me4Si as an internal standard and
dimethyl sulfoxide (DMSO-d6) and CDCl3 as the solvents. MS was performed on an 1100
LC/MSD spectrometer (Hewlett–Packard). Reactions were monitored by thin layer
chromatography (TLC) on GF/UV 254 plates, and the chromatograms were visualized under
UV light at 254 and 365 nm. After separation from the aqueous layers, the organic solvents
were dried over anhydrous sodium sulfate. All reagents were of reagent grade, and all
solvents were dried by standard methods before using. Column chromatography was carried
out on silica gel or alumina (200-300 mesh). Melting points were measured using a Mel-
TEMP II melting point apparatus and are uncorrected. Compounds d-f were synthesized as
previously described.[16, 17] The starting compound methyl 2-amino-4,5-dimethoxybenzoate
was commercially available.
The physical characteristics, MS, 1H-NMR and 13C-NMR data for all target compounds, are
reported in the Supporting Information.
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5.2. Cytotoxicity assay
First, 1×104 K562 and K562/A02 cells were seeded into 96-well plates in RPMI-1640 and
incubated for 24 h. In the assay of cytotoxicity, a graded dose of compounds diluted with
medium were added into the wells. In the assay of drug resistant modulation, 4 µM
concentrations of the target compounds were added into the wells, followed by numerous
concentrations of DOX. The cells were incubated for another 48 h in an atmosphere of 95%
air with 5% CO2 at 37 °C. Then, MTT was added directly to the cells. After additional
incubation for 4 h at 37 °C, the absorbance at 570 nm was read on a microplate reader
(Thermo Fisher Scientific). The IC50 values of the compounds for cytotoxicity were
calculated using GraphPad Prism 6.0 software from the dose–response curves.
5.3. MTT assay
K562/A02 cells were incubated in RPMI 1640 medium supplemented with 10% foetal bovine
serum at 37 °C in a 5% CO2 humidified atmosphere. K562/A02 cells were seeded into 96-
well plates at 1×104 cells per well. After 24 h of incubation, cells were treated with various
concentrations of DOX in the absence or presence of target compounds for 48 h in an
atmosphere of 95% air with 5% CO2 at 37 °C. Then, MTT was added directly to the cells.
After additional incubation for 4 h at 37 °C, the absorbance at 570 nm was read on a
microplate reader (Thermo Fisher Scientific). The IC50 values of the compounds for
cytotoxicity were calculated using GraphPad Prism 6.0 software from the dose–response
curves. Experiments were repeated three times independently.
5.4. Duration of the MDR reversal
First, 1~2×104 K562/A02 cells per well were plated into 96-well plates and cultured
overnight, then the cells were incubated for another 24 h with or without compound 19, VRP
or tariquidar at the concentration of 5 µM before being washed 0 or 3 times with growth
medium. Then, the cells were incubated for 0, 6, 12, or 24 h before addition of various
concentrations of DOX or the vehicle. The incubation was continued for 48 h prior to MTT
analysis. The IC50 values of the compounds for cytotoxicity were calculated by GraphPad
Prism 6.0 software from the dose-response curves.
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5.5. Accumulation of DOX
A laser confocal fluorescence microscope (FV 1000, Olympus, Japan) was used for in vitro
cell fluorescence imaging. K562 and K562/A02 cells were seeded into a laser co-focusing
dish at 1.5 ×105 cells/well. Various concentrations of compound 19 and VRP (10 μM, 2 μM,
and 0.2 μM) were preincubated with the cells for 60 min. Then, DOX was added into every
well and incubated for 90 min. The cells were fixed using 4% paraformaldehyde. The DOX
accumulation level was measured in photographs taken using a laser confocal fluorescence
microscope.
5.6. Rhodamine123 efflux assay
A laser confocal fluorescence microscope (FV 1000, Olympus, Japan) was used for in vitro
cell fluorescence imaging. K562 or K562/A02 cells were seeded into a laser co-focusing dish
at 1.5 ×105 cells/well and incubated with 5 µM Rh123 for 60 min before washing with ice-
cold PBS three times. Then, the cells were incubated with or without different concentrations
of compound 19 or VRP (10, 2, 0.2 µM) for another 90 min. The cells were fixed using 4%
paraformaldehyde (MeCHO). The mean fluorescence intensity of the maintained intracellular
Rh123 was assessed using a laser confocal fluorescence microscope.
Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China
(81673299) and the National Science and Technology Major Project of the Ministry of
Science and Technology of China (2018ZX09301034-004 and 2009ZX09102-033).
Figure 1.Structures of verapamil, dexverapamil, tariquidar and WK-X-34
Figure 2.Design of the target compounds.
Figure 3.Docking interaction of compound 19 with P-gp (PDB ID: 3G60)
Figure 4.EC50 for compound 19 in K562/A02 cells
Figure 5.The effects of the target compounds on the DOX accumulation in K562 or K562/A02 cells
which were detected and photographed under a laser confocal fluorescence microscope. A1: K562 cells
(DOX alone); A2: K562/A02 cells (DOX alone); B1-B3: K562/A02 cells DOX + numerous concentrations
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of compound 19 (B1: 10 µM, B2: 2 µM, B3: 0.2 µM); C1-C3: K562/A02 cells DOX + numerous
concentrations of VRP (C1: 10 µM, C2: 2 µM, C3: 0.2 µM).
Figure 6.The effects of the target compounds on the P-gp efflux function on intracellular Rh123 in K562
or K562/A02 cells which were detected and photographed under a laser confocal fluorescence microscope.
D: K562 cells (Rh123 alone); D1: K562/A02 cells (Rh123 alone); E1-E3: K562/A02 cells Rh123 +
numerous concentrations of compound 19 (E1: 10 µM, E2: 2 µM, E3: 0.2 µM); F1-F3: K562/A02 cells
Rh123 + numerous concentrations of VRP (F1: 10 µM, F2: 2 µM, F3: 0.2 µM).
Scheme 1: Synthesis of the target compounds. Reagents and conditions: (i) 3-bromoprop-1-yne, K2CO3, DMF,
reflux, 6 h; (ii) LiOH, 75% MeOH reflux, 3 h; (iii) substituted amine, HOBt, DIEA, DCM, EDCI, rt, 24 h; (iv)
K2CO3, acetonitrile, reflux, 17 h; (v) H2/Pd/C, DCM/EtOH, rt, 24 h; (vi) NaNO2, 50% AcOH, 0–5 °C, 30 min;
NaN3, 0–5 °C, 50 min; (vii), c1-c20 sodium ascorbate, CuSO4, 75% CH3OH, 24– 48 h.
Table 1.Cytotoxicity of compounds towards K562 and K562/A02 cell lines a
Table 2.DOX-resistance reversal activity of the target compounds 1–20 at 4 μM
concentration in K562/A02 cells a
Table 3.Effect of compound 19 on reversal of MDR in K562/A02 cells at different
concentrations a
Table 4.Duration of MDR reversal in K562/A02 cells after incubation and washout of verapamil,
tariquidar and compound 19
Conflicts of interest
There are no conflicts to declare.
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Table 1.Cytotoxicity of compounds towards K562 and K562/A02 cell lines a
Cytotoxicity IC50 (μM)
Cytotoxicity IC50 (μM)
Compounds
Compounds
K562
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
K562/A02
K562
K562/A02
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1
2
13
>100
14
>100
3
15
16
>100
4
>100
5
17
>100
6
18
>100
7
19
>100
8
20
70.13±6.4
30.77±1.94
3.092±2.1
61.51±3.4
28.8 ±1.2
107.3±1.9
9
VRP
Tariquidar
DOX
10
11
12
a
The IC50 values for the target compounds were determined by the MTT method. Each experiment
was carried out three times.
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Table 2.DOX-resistance reversal activity of the target compounds 1–20 at 4 μM
concentration in K562/A02 cells a
Compounds
IC50 of DOX (µM)
RF
Compounds
IC50 of DOX (µM)
RF
5
7.8
13.4
11.3
5.6
1
2
7.12 1.4
1.76 0.45
2.27 0.97
2.42 0.2
2.00 0.7
1.73 0.5
5.17 1.4
4.63 1.4
4.96 0.9
4.70 1.3
2.20 0.58
3.34
13
4.59 1.05
2.67 1.14
3.17
6.324 1.5
6.96 1.01
2.33 0.8
1.15 0.2
1.92 0.6
0.96 0.3
4.35 1.3
35.92 2.1
20.4
15.7
14.8
17.9
20.7
6.9
14
3
15
4
16
17
5.1
5
15.3
31.2
18.7
37.2
8.2
6
18
7
19
7.7
8
20
7.2
9
Tariquidar
VRP
Control b
7.6
10
11
12
16.2
1
10.7
Note: a The IC50 value was determined after exposure to a series of DOX concentrations combined with the
different target compounds at 4.0 μM using K562/A02 cells; reversal fold (RF, fold-change in drug sensitivity)
= (IC50 without inhibitor/(IC50 with inhibitor). b0.1% DMSO was added as a solvent control. Data were analysed
with GraphPad Prism 6.0 software and are presented as the mean ± SD of three independent tests.
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Table 3.Effect of compound 19 on reversal of MDR in K562/A02 cells at different
Compounds
IC50 of DOX (µM)
107.3±3.2
7.31±1.2
RF
1
None
VRP, 5 µM
14.7
77.2
64.3
38.3
5.2
Compound 19, 10 µM
Compound 19, 5 µM
Compound 19, 2.5 µM
Compound 19, 1.25 µM
Compound 19, 0.625 µM
Compound 19, 0.3125 µM
Compound 19, 0.156 µM
Compound 19, 0.078 µM
1.39±0.9
1.67±0.4
2.8±0.8
20.45±1.6
25.83±2.2
71.99±2.7
49.37±1.6
104.56±3.2
4.15
1.4
2.17
1.02
a
concentrations
a Reversal fold (RF), RF = (IC50 without modulator)/(IC50 with modulator). Each experiment was carried out
three times, and the values are displayed as the mean ± standard error of the mean.
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Table 4. Duration of MDR reversal in K562/A02 cells after incubation and washout of verapamil,
tariquidar and compound 19
Treatment
Schedule
IC50/DOX [μM] (RF)
VRP (5 μM)
Control (5 μM)
Compound19 (5μM)
2.45 ±0.2 (24.57)
5.32± 0.1(11.31)
9.98± 0.32(6.03)
21.99± 0.1(2.73)
33.67± 0.42(1.78)
Tariquidar (5 μM)
1.47± 0.11(40.96)
3.43± 0.3(17.55)
6.44 ± 0.21(9.35)
13.45± 0.1(4.47)
19.67± 0.32(3.06)
No wash
60.22±4.5(1)
6.54± 0.5(9.2)
Wash, 0 h
Wash, 6 h
Wash, 12 h
Wash, 24 h
nd
nd
nd
nd
33.6± 0.32(1.79)
nd
nd
nd
Numbers in parentheses indicate the reversal fold (RF), RF = (IC50 without modulator)/(IC50 with modulator).
Each experiment was carried out two to three times, and the values are displayed as the mean ± SD
nd: not identified.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.