Design, synthesis and biological evaluation of novel triazole-core reversal agents against P-glycoprotein-mediated multidrug resistance

Article abstract of DOI:10.1039/c6ra02405j

We designed and synthesized a novel series of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) inhibitors bearing a triazolphenethyl-tetrahydroisoquinoline scaffold through click chemistry. Then those synthesized compounds were tested on doxorubi

Full text of DOI:10.1039/c6ra02405j

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Design, synthesis and biological evaluation of novel triazole-core reversal agents
against P- glycoprotein-mediated multidrug resistance
Bo Zhang a, Tianxiao Zhao a, Jie Zhou a, Qianqian Qiu a, Yuxuan Dai a, Miaobo Pan a,
Wenlong Huanga a, b, * and Hai Qian a, b,
*
Abstract
We designed and synthesized a novel series of P-glycoprotein (P-gp)-mediated
multidrug resistance (MDR) inhibitors bearing a
triazolphenethyl-tetrahydroisoquinoline scaffold through click chemistry. Then those
synthesized compounds were tested on doxorubicin-resistant erythroleukemia
K562/A02 cells in a series of experiments. The result is that most of the synthesized
compounds exhibits more active MDR reversal activity than verapamil (VRP).
However, among them, compound 11 exhibits less cytotoxicity (IC50s > 100 µM), but
more potency than the known P-gp inhibitor WK-X-34 and VRP on increasing
anticancer drug accumulation in K562/A02 cells. Moreover, this compound can not
only persist longer chemo-sensitizing effect (>24h) than VRP (<6h) with reversibility,
but also significantly reverse MDR in a dose-dependent manner. Therefore,
considering the low intrinsic cytotoxicity but strong reversal activity in vitro,
compound 11 may be a promising lead with a potent and safe resistance modulator
in combinational cancer chemotherapy.

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1 Introduction
Tumor chemotherapy is a most important tool in the treatment of metastatic
cancer. However, drug resistance is regarded as the major barrier to successful
chemotherapy in cancer patients, a phenomenon known as multidrug resistance
[1,
(MDR)
2]. The resistance to therapy is caused by the over-expression of
membrane-associated transport proteins named ATP-binding cassette (ABC)
transporters including P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein
(BCRP/ABCG2), and multidrug resistance protein 1 (MRP⁄ABCC1), which has been
recognized as resulting in the increased efflux of chemotherapeutic drugs from
cancer cells [3, 4]. Among them, P-glycoprotein (P-gp) plays the most critical role in
MDR6 [5], which is a 170-kDa plasma membrane protein encoded by the MDR1 gene7
[6]. To overcome MDR, using specific inhibitors to direct inhibition of P-gp should be
regarded as a suitable and attractive approach [7]
.
During the past three decades, in order to reverse the MDR in cancer cells, three
[9]
,
generations of P-gp inhibitors have been developed such as Verapamil [8], VX-710
[10]
[11]
Zosuquidar (LY335979
and Tariquidar (XR9576)
(Fig. 1). However, because of
many factors, none of the P-gp inhibitors has been approved for clinical application,
such as unsatisfactory toxicity, low selectivity, poor potency or adverse
pharmacokinetic interaction with anticancer drugs [12, 13], which also lead to that, up
to now, no P-gp inhibitor has been approved by regulatory agencies [14]. Therefore,
the discovery of safe and effective P-gp inhibitors still remains to be a huge challenge
in this field.

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For designing novel P-gp inhibitors with high activity and low toxicity, we
selected the tetrahydroisoquinolinethylphenylamine-based P-gp inhibitor WK-X-34 as
a lead compound, which was designed and synthesized by researchers from the
University of Bonn through CADD (Computer-Aided Drug Design) and methodical
combinatorial chemistry. WK-X-34 is one of the third-generation of P-gp inhibitors,
which not only exhibits little competitive effect against combination therapy drugs,
but also shows relative high cytotoxicity (IC50 < 10 μM) [15]. Therefore, through click
chemistry method, amide group is replaced by 1, 2, 3-triazole ring, which is a
hydrophobic aromatic group, and can associate with biological targets through
hydrogen bonding (Fig. 2) [6, 17]. Click chemistry, which commonly employs the 1,
3-dipolar cycloaddition combined with the synthetic design of triazoles and
tetrazoles, has been widely applied in drug discovery [18]. On the basis of principles
above, we designed and synthesized a series of P-gp-mediated MDR reversal agents.
Herein, we report the biological activity of newly synthesized P-gp-mediated MDR
reversal agent.
2 Chemistry
Compounds 1-12 were prepared starting from 2-nitrophenol and 3,
4-dimethoxyphenethylamine, and the synthetic routes of target compounds were
outlined in Scheme 1. Firstly, 2-nitrophenol and 3-bromo-1-propyne were refluxed in
the mixture of acetone and potassium carbonate for 4h affording compound a.
Secondly, compound a reacted with ammonium chloride in the presence of iron

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powder in 80% ethanol affording compound b, which then reacted with freshly
formed aromatic acyl chloride and triethylamine in dry dichloromethane to give
compounds c1-c12. Compounds d and e were synthesized according to literature
procedures with minor modification [16]. Subsequently, compounds e reacted with
sodium azide affording compound f. Last, compound f reacted with diverse alkyne
c1-c12 through click chemistry to provide compounds 1-12. The structures of target
compounds obtained were listed in Table 1.
3 Results and discussion
3.1 Cytotoxicity of the target compounds
To identify ideal P-gp inhibitors reversing MDR at non-toxic concentrations, MTT
(3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay was adopted to
evaluate the intrinsic cytotoxicity of the target compounds against the human
erythroleukemia K562 cells and the adriamycin (ADM)-resistant K562/A02 cells,
which shows MDR due to P-gp overexpression (induced by ADM) and has been
proved as useful in the quantification and characterization of MDR reversal by
modulators of the MDR phenotype [19, 20]. We chose verapamil (VRP) as the positive
control, which is a well-known classical P-gp inhibitor. As shown in Table 1, the
majority of the target compounds displays low intrinsic cytotoxicity since their IC50
values are at a high micromolar level. As expected, K562/A02 cells are significantly
resistant to ADR: K562/A02 (IC50 for ADM of 71.04 ± 3.2 µM) displaying about
151.9-fold higher than K562 cells (IC50 of 0.47 ± 0.03 µM). In contrast, compound 5

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and 7 is found to exhibit high toxicity (IC50 < 25 µM) in both cell lines, VRP has weak
cytotoxic effect toward K562 and K562/A02 cells with IC50 values 41.6 and 37.2 µM
and WK-X-34 displaying a high level of toxicity toward K562 cells (IC50 of 6.9±1.9µM)
but a weak toxicity toward K562/A02 cells (IC50 of 33.7±2.7µM). Based on the IC50s,
most of target compounds possess low cytotoxicity in tested cell lines and are
suitable candidates for the development of P-gp inhibitors.
3.2 Effects of the target compounds on reversing ADM resistance in K562/A02 cells
Based on the preliminary experiment, most of target compounds possess low
cytotoxicity in tested cell lines. The reversal effects of target compounds on the MDR
phenotype have been examined in K562/A02 cells at 0.5µM-concentrations by MTT
assay. As the results summarized in Table 2, anticancer drug ADM alone displays little
inhibitory effect on the survival of K562/A02 cells (IC50 of 71.04 ± 3.2 µM). However,
the reversal fold (RF) data indicates that all target compounds and VRP increase the
inhibitory effect of different doses. It reveals that all of the target compounds
possess MDR reversal activity. Moreover, most of the target compounds exhibits
more active MDR reversal activity than the classical P-gp inhibitor VRP, when
co-administered with ADM at the same condition. Notably, compound 11 shows the
strongest reversal activity among the synthesized compounds (IC50 of 1.76 ± 0.3 µM).
3.3 Chemo-sensitizing effect of target compounds
Based on the results of preliminary experiments, we selected the most potent

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compound 11 for further study. To further investigate the reversal potency and dose–
response effects, the cytotoxicity of ADM against K562/A02 cells
(P-gp-overexpression) was evaluated in the presence of the compound 11 at various
concentrations (0.10 µM, 0.25 µM, 0.50 µM, 1.00 µM, 2.50 µM, 5.00 µM, 10.00 µM,
20.00 µM) by MTT assay [21]. As the results summarized in Table 3, compound 11
showed apparent dose dependent activity and still exhibited potent MDR reversal
activity ( RF = 4.6 ) when the concentration decreased to 1.00 µM. Additionally, the
EC50 value of compound 11 was 488.5 ± 71.2 nM (Fig. 3), which was analyzed by
GraphPad Prism 5.0 software from the dose–response curves. Those results indicated
that compound 11 significant potentiated the cytotoxicity of ADM in K562/A02 cells
in a dose-dependent manner.
3.4 Duration of MDR reversal effect of compound 11 toward ADM in K562/A02 cells
It is known that an ideal P-gp inhibitor should possess a relatively long duration
of action with reversibility for safe and effective therapy of P-gp-mediated MDR
cancers [22]. Therefore, we used VRP and WK-X-34 as positive controls to evaluate the
duration of MDR reversal effect of compound 11. The experiment was executed as
previous report with minor modification [21]. K562/A02 cells were incubated with 5
µM of compound 11, VRP and WK-X-34 for 24h, and then we washed compounds
with PBS (phosphate buffered saline). At different time points (0, 6, 12 or 24h), after
removing the compounds, we added ADM in various concentrations to the culture,
and incubated for additional 48 h. The results, examined by MTT assay, were shown

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in Table 4: after pre-incubation with VRP, WK-X-34 and compound 11, the IC50s of
ADM towards K562/A02 cells were 19.32 µM, 2.32 µM, 2.11 µM respectively (no
wash group). The MDR-reversing effect of VRP (RF = 1.6) waned immediately after its
removal from the medium and totally disappeared after 6h. In contrast, compound
11 and WK-X-34 showed a very strong reversal activity even after removed from the
medium for 24h, and the IC50s of ADM were 32.41 µM (RF = 2.5) and 12.91µM (RF =
6.2), respectively. Moreover, compound 11 displayed potent chemo-sensitizing effect
and persisted for longer time compared with the positive control VRP. The data also
indicated that the MDR reversal effect of compound 11 towards K562/A02 cells was
reversible.
3.5 Accumulation of ADM
Furthermore, in order to investigate the compound 11 can reverse
P-gp-mediated drug efflux, we selected ADM as a fluorescent probe, which was a
fluorescent substrate of P-gp and could be used to monitor drug accumulation in
[23]
cells
,
and measured the fluorescence intensity of ADM through
spectrofluorometry according to the previous report with minor modification. As
shown in Fig. 4, the P-gp inhibitor VRP and WK-X-34 were selected as positive
controls. The ADM-sensitive K562 cells retained most of the fluorescence ADM, while
the P-gp-overexpressing K562/A02 cells showed comparatively low intracellular level
of ADM. The fluorescence intensity of ADM in K562/A02 cells, treated with
compound 11, VRP or WK-X-34, was obviously increased in a dose-dependent

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manner. In comparison, Compound 11 showed a higher level of ADM accumulation
than VRP at the same dose, and has similar reversal activity compared with
WK-X-34.The results indicated that compound 11 exhibited similar potency with
WK-X-34 at different concentrations and more potent than VRP in inhibiting the ADM
transport activity of P-gp.
3.6 Inhibitory effect of compound 11 on P-gp efflux function
In order to further validate our assumption, we selected rhodamine-123 (Rh123)
as another fluorescent probe, which was a well-known fluorescent substrate of P-gp
and could be used to investigate whether compound 11 could reverse P-gp-mediated
drug efflux by targeting P-gp. It is known that P-gp transporter can rapidly reduce
Rh123 in the intracellular accumulation through an increase of Rh123 efflux from
cells, which overexpresses on the adriamycin-selected K562/A02 cells. The level of
Rh123 was measured by monitoring its fluorescence intensity through flow
cytometry assay according to the method described in literatures with minor
modification [24, 25]. As shown in Fig. 5, the P-gp inhibitor VRP and WK-X-34 were
selected as positive controls. The adriamycin-sensitive K562 cells retained most of
the fluorescence Rh123, while the P-gp-overexpressing K562/A02 cells showed
comparatively low intracellular level of Rh123. In comparison, treated with
compound 11, VRP or WK-X-34, the fluorescence intensity of Rh123 in K562/A02
cells was obviously increased in a dose-dependent manner. Particularly, compound
11 showed a higher level of Rh123 accumulation than VRP at the same dose, and had

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similar reversal activity compared with WK-X-34. The results indicated that
compound 11 was targeting p-gp and could effectively block the drug efflux.
4 Conclusions and Future Directions
In summary, we selected WK-X-34 as a lead compound, and twelve compounds
were newly designed and synthesized based on the click chemistry. All synthesized
compounds were evaluated in vitro for MDR chemo-sensitizing effects by using the
classical P-gp inhibitor VRP as a positive control. Most of target compounds
possessed low cytotoxicity and exhibited more active MDR reversal activity than VRP,
especially for Compound 11, which showed the strongest reversal activity and could
effectively prevent the ADM efflux function of P-gp and increased the accumulation
of ADM in K562/A02 cells. Therefore, compound 11 could be served as a promising
candidate to develop P-gp-mediated MDR reversal modulator in cancer
chemotherapy and further intensive investigations are still in progress.
5 Experimental section
5.1 General chemistry
All reagents and solvents were reagent grade and were used without further
purification unless otherwise stated. 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, which was uncorrected. All of the target
1
compounds were analyzed by H NMR, 13C NMR (Bruker ACF-300Q, 300 MHz), MS

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(1100 LC/MSD spectrometer; Hewlett–Packard) and IR (Nicolet Impact 410);
Thin-layer chromatography (TLC) was performed on GF/UV 254 plates and the
chromato-grams were visualized under UV light at 254 and 365 nm. Compounds d
and e were prepared as previously described [16]. The starting compound
2-nitrophenol was commercially available.
5.2 Synthesis of 1-Nitro-2-(prop-2-yn-1-yloxy) benzene (a)
The mixture of 2-nitrophenol (11.2 g, 80 mmol), 3-bromoprop-1-yne (9.52 g, 80
mmol), and potassium carbonate (22.1 g, 160 mmol) in acetone (120 mL) was heated
to reflux for 3 h. The reaction mixture was cooled to 0 °C followed by filtration and
concentration to afford 12.6 g compound a. Yield: 89.1%. Yellow powder, m.p.: 66–
68 °C.
5.3 Synthesis of 2-(Prop-2-yn-1-yloxy) aniline (b)
The mixture of a (12.4 g, 70 mmol), ammonium chloride (18.7 g, 350 mmol) and
iron powder (11.7 g, 210 mmol) in 80% ethanol (150 mL) was heated to reflux for 3.5
h. After cooling to room temperature, the mixture was adjusted with sodium
carbonate to PH = 7–8, filtered with Celite, and concentrated under vacuum to afford
a brown residue. Dichloromethane (50 mL) was added into the residue and the
resulted solution was washed by saturated aqueous Na2CO3 (30 mL) and brine (30 mL)
in sequence. The organic layer was dried by anhydrous Na2SO4. The solvent was
evaporated to afford 8.40 g compound b as brown oil. Yield: 81.7%.

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5.4 General procedure for the preparation of c1–c12
Synthesis of c3, c6, c8: to the solution of compound b (0.74g, 5 mmol) and
triethylamine (0.84g, 6 mmol) in dichloromethane (20 mL), heterocyclic aromatic acyl
chloride (5 mmol) in dichloromethane (15 mL) was added at 0 °C, and then, the
mixture was stirred at room temperature for 24 h. The reaction solution was washed
by 3% hydrochloric acid (2 × 20 mL), saturated aqueous Na2CO3 (20 mL), and brine
(20 mL) in sequence. The organic layer was dried by anhydrous Na2SO4. The solvent
was evaporated to afford the desired product c3, c6 and c8. Synthesis of other
compounds: to the solution of compound b (0.74g, 5 mmol) and heterocyclic
aromatic formic acid (5 mmol) in dichloromethane (20 mL), EDC·HCl (1.44 g , 7.5
mmol) in dichloromethane (15 mL) was added, and then, the mixture was stirred at
room temperature for 24 h. The reaction solution was washed by 3% hydrochloric
acid (2 × 20 mL), saturated aqueous Na2CO3 (20 mL), and brine (20 mL) in sequence.
The organic layer was dried by anhydrous Na2SO4. Then, filtered and evaporated to
afford the desired product compound c1–c12. Yield: 45.7%–86.3%
5.5
Synthesis
of
2-(4-Azidophenethyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (f)
Compound e (1.9 g, 6.1 mmol) was dissolved in 50% acetic acid (30 mL). To this
solution, sodium nitrite (0.6 g, 8.5 mmol) was slowly added at -5 to 0 °C within 30
min. The solution was vigorously stirred at 0–5 °C for 50 min. Sodium azide (0.6 g, 9.2

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mmol) was batch added into the reaction mixture at 0–5 °C. The resulting solution
was stirred at 0–5 °C for 1 h followed by diluting with ice water (200 mL) and
extracting with EtOAc (3 × 100 mL). The combined organic layer was washed with
water (2 × 60 mL), saturated aqueous NaHCO3 (2 × 60 mL) and brine (2 × 50 mL),
dried over anhydrous Na2SO4, filtered and concentrated to afford 1.2 g pink solid.
Yield 58.2%. m.p.: 68–70 °C
5.6 General procedure for the preparation of compounds 1–12
To the solution of c1–c12 (1 mmol) and f (1 mmol) in 75% methanol (40 mL),
ascorbate sodium (30 mg) and CuSO4 (10 mg) were added, respectively. The reaction
solution was stirred at room temperature for 24–48 h. After filtration, the solvent
was evaporated and the crude product was purified by silica gel column
chromatography using a mixture of ethylacetate/ Methanol (15:1, v/v) as eluent to
give the desire product with high purity. The purity of each tested compounds (>95%)
was determined on a Waters UPLC/MS instrument using a Waters C18 column (1.7
μm 2.1× 50 mm, with a flow rate of 0.2 mL/min and detection at 254 nm) employing
a 10–90% acetonitrile/water/0.1% formic acid gradient.
5.6.1
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)picolinamide (1)
1
Yield 62.3%; white powder; mp: 164-165 °C. H NMR (300 MHz, DMSO-d6) δ:

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10.52 (s, 1H, CONH), 8.94 (s, 1H, NCH=C), 8.58 (d, J = 3.2 Hz, 1H, ArH), 8.45 (d, J = 7.5
Hz, 1H, ArH), 8.17 (d, J = 7.4 Hz, 1H, ArH), 8.03-8.11 (m, 1H, ArH), 7.78 (d, J = 7.8 Hz,
2H, ArH), 7.63 (d, J = 5.1 Hz, 1H, ArH), 7.49 (d, J = 7.7 Hz, 2H, ArH), 7.39 (d, J = 7.8 Hz,
1H, ArH), 7.15-7.05 (m, 2H, ArH), 6.64 (d, J = 7.5 Hz, 2H, ArH), 5.45 (s, 2H, OCH2), 3.70
(s, 6H, 2 × OCH3), 3.32 (s, 2H, ArCH2N), 2.90-2.71 (m, 8H, 4 × CH2); 13C NMR (75 MHz,
DMSO-d6) δ: 152.7, 151.3, 148.2, 147.6, 146.7, 144.4, 139.4, 137.5, 134.8, 134.8,
134.0, 128.2 127.7, 127.7, 126.8, 126.7, 126.5, 124.2, 122.1, 121.2, 120.3, 119.1,
112.8, 111.4, 108.0, 72.3, 59.7, 58.0, 56.3, 56.1, 33.4, 27.3; ESI-MS m/z: 590.7 [M+H]+;
IR (KBr, cm-1) υ: 3321.63, 1682.10, 1253.90 (-CONH-), 2926.25, 1227.37 (-OCH3),
1601.04,
1533.16, 1519.15, 1454.96, 759.28, 689.13.
5.6.2
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)isonicotinamide (2)
1
Yield 72.9%; white powder; mp: 112-114 °C; H NMR (300 MHz, DMSO-d6) δ:
9.84 (s, 1H, CONH), 8.82 (s, 2H, ArH), 7.86 (s, 2H, ArH, NCH=C), 7.73 (d, J = 8.2 Hz,
4H,ArH), 7.48 (d, J = 8.3 Hz, 2H, ArH), 7.38 (d, J = 8.1 Hz, 1H, ArH), 7.25 (t, J = 7.1 Hz,
1H, ArH),7.04 (d, J = 7.5 Hz, 1H, ArH), 6.64 (d, J = 8.0 Hz, 2H, ArH), 5.34 (s, 2H, OCH2),
3.70 (s, 6H, 2 × OCH3), 3.55 (s, 2H, ArCH2N), 2.93-2.70 (m, 8H, 4 × CH2); 13C NMR (75
MHz, DMSO-d6) δ: 164.7, 152.7, 149.7, 148.2, 146.7, 144.4, 140.8, 139.4, 134.8,
134.8, 134.0, 128.2, 127.7, 127.7, 126.8, 126.5, 124.2, 121.7, 121.7, 121.2, 120.3,

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119.1, 112.8, 111.4, 108.3, 72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z:
591.4 [M+H]+; IR (KBr, cm-1) υ: 3410.09, 1666.55, 1257.70 (-CONH-), 2938.05,
1226.55 (-OCH3), 1599.35, 1540.60, 1519.05, 1452.51, 1129.59, 750.87.
5.6.3
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)furan-2-carboxamide (3)
1
Yield 56.1%; brown powder; mp: 136-138 °C; H NMR (300 MHz, DMSO-d6) δ:
9.16 (s, 1H, CONH), 8.90 (s, 1H, NCH=C), 7.97 (d, J = 7.9 Hz, 1H, ArH), 7.88 (s, 1H, ArH),
7.82 (d, J = 8.2 Hz, 2H, ArH), 7.52 (d, J = 8.3 Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 1H, ArH),
7.29 (d, J = 3.3 Hz, 1H, ArH), 7.17 (t, J = 6.7 Hz, 1H, ArH), 7.01 (t, J = 7.7 Hz, 1H, ArH),
6.74 (s, 1H, ArH), 6.70-6.68 (m, 2H, ArH), 5, 39 (s, 2H, OCH2), 3.98 (s, 2H, ArCH2N),
3.72 (s, 6H, 2 × OCH3), 3.33-2.73 (m, 8H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6) δ:
162.7, 152.7, 148.2, 148.2, 146.7, 144.4, 143.8, 139.4, 134.8, 134.8, 134.0, 128.2,
127.7, 127.7, 126.8, 126.5, 124.2, 121.2, 120.3, 119.1, 115.3, 112.8, 111.7, 111.4,
108.3, 72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3. ESI-MS m/z: 580.9 [M+H]+; IR (KBr,
cm-1) υ: 3404.13, 1655.59, 1254.53 (-CONH-), 2932.15, 1227.88 (-OCH3), 1583.32,
1538.77, 1518.35, 1452.78, 1130.76, 755.12.
5.6.4
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)furan-3-carboxamide (4)

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1
Yield 59.0%; brown powder; mp: 143-145 °C; H NMR (300 MHz, DMSO-d6)
δ:9.22 (s, 1H, CONH), 8.83 (s, 1H, NCH=C), 8.38 (s, 1H, ArH), 7.78-7.72 (m, 4H, ArH),
7.49 (d, J = 8.4 Hz, 2H, ArH), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.19 (t, J = 7.2 Hz, 1H, ArH),
7.03-6.95 (m, 2H, ArH), 6.64 (d, J = 7.7 Hz, 2H, ArH), 5.32 (s, 2H, OCH2), 3.69 (s, 6H, 2
× OCH3), 3.55 (s, 2H, ArCH2N), 2.93-2.71 (m, 8H, 4 × CH2); 13C NMR (75 MHz,
DMSO-d6) δ: 164.7, 152.7, 151.1, 148.2, 146.7, 144.8, 144.4, 139.4, 134.8, 134.8, 134,
128.8, 128.2, 127.7, 127.7, 126.8, 126.5, 124.2, 121.2, 120.3, 119.1, 112.3, 111.4,
108.3, 107.9, 72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z: 580.3 [M+H]+;
IR (KBr, cm-1) υ: 3115.04, 1665.08, 1257.55 (-CONH-), 2938.05, 1227.76 (-OCH3),
1599.26, 1542.37, 1452.95, 1129.80, 754.01.
5.6.5
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)-1H-indole-3-carboxamide (5)
1
Yield 49.6%; brown powder; mp: 157-159 °C; H NMR (300 MHz, DMSO-d6) δ:
11.82 (s, 1H, NH), 8.90 (s, 1H, CONH), 8.85 (s, 1H, NCH=C), 8.17-8.13 (m, 2H, ArH),
7.94 (d, J = 7.8 Hz, 1H, ArH), 7.73 (d, J = 8.1 Hz, 2H, ArH), 7.48 (s, 1H, ArH), 7.46-7.43
(m, 2H, ArH), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.16-6.94 (m, 4H, ArH), 6.65 (d, J = 7.4 Hz,
2H, ArH), 5.37 (s, 2H, OCH2), 3.70 (s, 6H, 2 × OCH3), 3.35 (s, 2H, ArCH2N), 3.18-2.72
(m, 8H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6) δ: 164.7, 152.7, 148.2, 146.7, 144.4,
139.4, 138.6, 134.8, 134.8, 134.0, 130.7, 128.2, 128.2, 127.7, 126.8, 126.5, 124.2,
121.8, 121.7, 121.2, 120.3, 119.1, 112.8, 112.0, 111.4, 111.1, 108.3, 72.3, 59.7, 58.0,

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56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z: 629.4 [M+H]+; IR (KBr, cm-1) υ: 3439.53,
1654.51, 1244.75 (-CONH-), 2938.05, 1229.02 (-OCH3), 1518.43, 1449.65, 1129.30,
747.89
5.6.6
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)benzofuran-2-carboxamide (6)
1
Yield 43.9%; brown powder; mp: 185-186 °C; H NMR (300 MHz, DMSO-d6) δ:
9.55 (s, 1H, CONH), 8.93 (s, 1H, NCH=C), 8.00 (s, 1H, ArH), 7.65 (s, 4H, ArH), 7.54 (m,
4H, ArH), 7.05 (s, 4H, ArH), 6.64 (s, 2H, ArH), 5.40 (s, 2H, OCH2), 3.69 (s, 6H, 2 × OCH3),
2.90 (s, 2H, ArCH2N), 2.71 (m, 8H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6) δ: 162.7,
157.2, 152.7, 150.0, 148.2, 146.7, 144.4, 139.4, 134.8, 134.8, 134.0, 128.2, 127.7,
127.7, 126.9, 126.8, 126.5, 124.7, 124.2, 123.3, 121.2, 120.9, 120.3, 119.1, 112.8,
111.5, 111.4, 108.9, 108.3, 72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z:
630.5 [M+H]+; IR (KBr, cm-1) υ: 3392.40, 1681.31, 1255.42 (-CONH-), 2908.55,
1224.85 (-OCH3), 1538.34, 1518.73, 1454.83, 1134.29, 753.85.
5.6.7
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxami
de (7)
Yield 61.9%; gray powder; mp: 139-141 °C; 1H NMR (300 MHz, DMSO-d6) δ:10.03

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(s, 1H, CONH), 8.92 (s, 1H,NCH=C), 8.87 (s, 1H, CH = C), 8.01 (d, J = 7.6 Hz, 1H, ArH),
7.78-7.69 (m, 4H, ArH), 7.46 (d, J = 8.2 Hz, 2H, ArH), 7.38-7.35 (m, 2H, ArH), 7.24 (t, J
= 7.5, 1H, ArH), 7.06 (t, J = 7.6, 1H, ArH), 6.64 (d, J = 6.3 Hz, 2H, ArH), 5.33 (s, 2H,
OCH2), 4.00 (s, 3H, OCH3), 3.70 (s, 6H, 2 × OCH3), 3,46 (s, 2H, ArCH2N), 2.94-2.71 (m,
8H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6) δ: 174.5, 163.0, 156.7, 152.7, 148.2, 147.7,
146.7, 144.4, 139.4, 134.8, 134.8, 134.8, 134.0, 128.2, 127.7, 127.7, 126.8, 126.5,
124.2, 124.1, 121.2, 120.3, 119.1, 117.1, 112.8, 111.4, 108.3, 95.6, 72.3, 59.7, 58.0,
56.3, 56.1, 56.1, 55.9, 54.0, 46.0, 33.4, 27.3; ESI-MS m/z: 686.7 [M+H]+; IR (KBr, cm-1)
υ: 3445.43, 1670.84, 1270.60 (-CONH-), 2926.25, 1227.56 (-OCH3), 1519.42, 1452.95,
1118.97, 748.91.
5.6.8
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)thiophene-2-carboxamide (8)
1
Yield 76.9%; brown powder; mp: 160-162 °C; H NMR (300 MHz, DMSO-d6)
δ:9.57 (s, 1H, CONH), 8.84 (s, 1H, NCH=C), 7.97 (d, J = 2.9 Hz, 1H, ArH), 7.83 (d, J = 4.9
Hz, 1H,ArH), 7.77 (d, J = 8.2 Hz, 2H, ArH), 7.70 (d, J = 7.7 Hz, 1H, ArH), 7.50 (d, J = 8.3
Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 1H, ArH), 7.24-7.18 (m, 2H, ArH), 7.03 (d, J = 7.5 Hz,
1H, ArH), 6.70 (d, J = 9.4 Hz, 2H, ArH),5.35 (s, 2H, OCH2), 3.82 (s, 2H, ArCH2N), 3.72 (s,
6H, 2 × OCH3), 3.00-2.83 (m, 8H, 4 × CH2); 13C NMR (75 MHz, DMSO-d6) δ: 161.8,
152.7, 148.2, 146.7, 144.4, 139.4, 139.4, 134.8, 134.8, 134.0, 131.9, 130.3, 129.0,
128.2, 127.7, 127.7, 126.8, 126.5, 124.2, 121.2, 120.3, 119.1, 112.8, 111.4, 108.3,

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72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z: 596.3 [M+H]+; IR (KBr, cm-1)
υ: 3410.03, 1650.26, 1255.30 (-CONH-), 2926.25, 1228.89 (-OCH3), 1539.05, 1517.97,
1355.07, 1122.56, 743.12, 721.79.
5.6.9
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)thiophene-3-carboxamide (9)
1
Yield 78.4%; brown powder; mp: 84-86 °C; H NMR (300 MHz, DMSO-d6) δ:9.35
(s, 1H, CONH), 8.82 (s, 1H, NCH=C), 8.34 (s, 1H, ArH), 7.78-7.72 (m, 3H, ArH),
7.65-7.59 (m, 2H, ArH), 7.48 (d, J = 8.2 Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 1H, ArH), 7.20
(t, J = 7.1 Hz, 1H, ArH), 7.00 (t, J = 7.5 Hz, 1H, ArH), 6.64 (d, J = 7.6 Hz, 2H, ArH), 5.34
(s, 2H, OCH2), 3.70 (s, 6H, 2 × OCH3), 3.40 (s, 4H, 2 × CH2), 3.71 (s, 6H, 3 × CH2); 13C
NMR (75 MHz, DMSO-d6) δ: 174.7, 152.7, 148.2, 146.7, 144.4, 139.4, 136.3, 134.8,
134.0, 129.0, 128.2, 127.7, 127.7, 127.6, 126.8, 126.6, 126.5, 124.2, 121.2, 120.3,
119.1, 112.8, 111.4, 108.3, 72.3, 59.7, 58.0, 56.3, 56.1, 56.1, 33.4, 27.3; ESI-MS m/z:
597.1 [M+H]+; IR (KBr, cm-1) υ: 3091.45, 1658.70, 1257.22 (-CONH-), 2926.25,
1227.88 (-OCH3), 1533.09, 1517.75, 1128.25, 846.12, 751.56.
5.6.10
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)-3-methylthiophene-2-carboxamide (10)
1
Yield 52.4%; brown powder; mp: 163-165 °C; H NMR (300 MHz, DMSO-d6) δ:

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8.92 (s, 1H, CONH), 8.83 (s, 1H, NCH=C), 8.10 (d, J = 7.5 Hz, 1H, ArH), 7.78 (d, J = 7.3
Hz, 2H, ArH), 7.66 (d, J = 4.0 Hz, 1H, ArH), 7.49 (d, J = 7.1 Hz, 2H, ArH), 7.35 (d, J = 7.4
Hz, 1H, ArH), 7.17 (s, 1H, ArH), 7.04-6.99 (m, 2H, ArH), 6.64 (d, J = 6.9 Hz, 2H, ArH),
5.35 (s, 2H, OCH2), 3.93 (s, 6H, 2 × OCH3), 3.55 (s, 2H, CH2), 2.91 (s, 2H, CH2), 2.71 (s,
6H, 3 × CH2), 2.36 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ: 161.8, 152.7, 148.2,
146.7, 144.4, 139.4, 136.3, 135.0, 134.8, 134.8, 134.0, 129.2, 128.2, 127.7, 127.7,
126.8, 126.5, 124.5, 124.2, 121.2, 120.3, 119.1, 112.8, 111.4, 108.3, 72.3, 59.7, 58.0,
56.3, 56.1, 56.1, 33.4, 27.3, 13.7; ESI-MS m/z: 610.8 [M+H]+; IR (KBr, cm-1) υ: 3427.73,
1638.00, 1251.86 (-CONH-), 2926.25, 1227.55 (-OCH3), 1536.27, 1518.12, 1125.48,
756.40, 743.31.
5.6.11
N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-1H-1,2,
3-triazol-4-yl)methoxy)phenyl)-5-methylthiophene-2-carboxamide (11)
1
Yield 50.1%; brown powder; mp: 146-147 °C; H NMR (300 MHz, DMSO-d6) δ:
9.40 (s, 1H, CONH), 8.81 (s, 1H, NCH=C), 7.74 (s, 4H, ArH), 7.49 (s, 2H, ArH), 7.34 (m,
1H, ArH), 7.20 (m, 1H, ArH), 7.01 (m, 1H, ArH), 6.88 (s, 1H, ArH), 6.65 (m, 2H, ArH),
5.34 (s, 2H, OCH2), 3.70 (s, 6H, 2 × OCH3), 3.56 (s, 2H, CH2), 2.90 (s, 2H, CH2), 2.71 (s,
6H, 3 × CH2), 2.46 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ: 161.8, 152.9, 152.7,
148.2, 146.7, 144.4, 139.4, 135.3, 134.8, 134.8, 134.0, 129.5, 128.5, 128.2, 127.7,
127.7, 126.8, 126.5, 124.2, 121.2, 120.3, 119.1, 112.8, 111.4, 108.3, 72.3, 59.7, 58.0,
56.3, 56.1, 33.4, 27.3, 14.5; ESI-MS m/z: 610.7 [M+H]+; IR (KBr, cm-1) υ: 3415.93,

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1655.91, 1254.81 (-CONH-), 2932.15, 1227.35 (-OCH3), 1521.96, 1450.91, 1128.87,
738.78.
5.6.12
5-chloro-N-(2-((1-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)pheny
l)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)thiophene-2-carboxamide (12)
1
Yield 79.4%; brown powder; mp: 128-130 °C; H NMR (300 MHz, DMSO-d6) δ:
9.73 (s, 1H, CONH), 8.80 (s, 1H, NCH=C), 7.86 (d, J = 2.8 Hz, 1H, ArH), 7.74 (d, J = 7.6
Hz, 2H, ArH), 7.61 (d, J = 7.5 Hz, 1H, ArH), 7.48 (d, J = 7.6 Hz, 2H, ArH), 7.37 (d, J = 7.9
Hz, 1H, ArH), 7.23 (t, J = 9.0 Hz, 2H, ArH), 7.02 (t, J = 7.5 Hz, 1H, ArH), 6.64 (d, J = 7.2
Hz, 2H, ArH), 5.33 (s, 2H, OCH2), 3.70 (s, 6H, 2 × OCH3), 3.56 (s, 2H, CH2), 2.90 (s, 2H,
CH2), 2.71 (s, 6H, 3 × CH2); 13C NMR (75 MHz, DMSO-d6) δ: 161.8, 152.7, 148.2, 146.7,
144.4, 140.7, 139.4, 138.3, 135.9, 134.8, 134.8, 134.0, 129.8, 128.2, 127.7, 127.7,
126.8, 126.5, 124.2, 121.2, 120.3, 119.1, 112.8, 111.4, 108.3, 72.3, 59.7, 58.0, 56.3,
56.1, 56.1, 33.4, 27.3; ESI-MS m/z: 630.7 [M+H]+; IR (KBr, cm-1) υ: 3345.13, 1657.42,
1255.05 (-CONH-), 2926.25, 1229.30 (-OCH3), 1541.17, 1518.72, 1453.45, 1130.33,
745.36.
5.9 Materials and methods
5.9.1 Materials
Adriamycin (ADM), verapamil (VRP), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT), and DMSO were purchased from Sigma-Aldrich (St. Louis,

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MO, USA). WK-X-34 with high purity was synthesized by our laboratory before [20]. All
other chemicals were molecular biology grade and obtained from Sigma-Aldrich or
Thermo Fisher Scientific (Waltham, MA, USA).
5.9.2 Cell lines and cell culture
Human leukemia cell line K562 and its adriamycin-selected P-gp-overexpressing
subline K562/A02 were kindly provided by Dr. Dr. Yun-Man Li (Department of
Physiology, China Pharmaceutical University, Nanjing, China). The cell lines were
grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and incubated
at 37 °C in a humidified incubator with 5% CO2 in air growth. To maintain MDR
phenotype, 1 mg/mL adriamycin was added to K562/A02 cultures and maintained in
drug-free medium for 2 weeks before use. All experiments were performed with cells
in exponential growth. To maintain MDR phenotype, 1 mg/mL adriamycin was added
to the K562/A02 cultures and maintained in a drug-free medium for 2 weeks before
use.
5.9.3 MTT assay
K562/A02 cells were incubated in RPMI 1640 medium supplemented with 10%
fetal bovine serum at 37 °C in a 5% CO2 humidified atmosphere. K562/A02 cells (1-2 ×
104cells per well) were seeded in 96-well plates. After 24 h incubation, cells were
treated with various concentrations of ADM in absence or presence of target
compounds for 48 h in an atmosphere of 95% air with 5% CO2 at 37 °C. Then, MTT

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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 by GraphPad Prism
5.0 software from the dose–response curves. Experiments were conducted in
triplicates and repeated three times independently.
5.9.4 Duration of the MDR reversal
1-2 × 104 K562/A02 cells per well were plated in 96-well plates and cultured
overnight, then the cells were incubated for another 24 h with or without compound
11, VRP , WK-X-34 or PBS at the concentration of 5µM before being washed 0 or 3
times with growth medium. Then, the cells were incubated for 0, 6, 12 or 24 h before
the addition of various concentrations of ADM or vehicle. The incubation was
continued for 48 h prior to the MTT analysis. The IC50 values of the compounds for
cytotoxicity were calculated by GraphPad Prism 5.0 software from the dose–
response curves.
5.9.5 Accumulation of ADM
ADM accumulation assay was performed according to the reported procedures
with minor modification [26]. In brief, 2 × 105 cells of K562 and K562/A02 were
incubated with 20 µM ADM and different concentrations of compound 11 for 2.5 h at
37 °C. 0.1% DMSO was used as a negative control. VRP and WK-X-34 were used as
positive controls. After incubation, the cells were washed with cold PBS and lysed

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with lysis buffer (0.75 M HCl, 0.2% Triton-X100 in isopropanol). The fluorescence
level of ADM in the lysate was determined by fluorescence spectrophotometer
(RF-5301 PC; Shimadzu) using an excitation and an emission wavelength pair of 460
and 610 nm. Accumulation was expressed as ADM fluorescent intensity (FI) per 2 ×
105 cells. All incubations were carried out in triplicate and three independent
experiments were performed.
5.9.6. Rhodamine123 efflux assay
K562 or K562/A02 cells were seeded into 24-well plates at 1.5 × 105/well and
incubated with 5 µM Rh123 for 60 min before washing with ice PBS for three times.
Then the cells were incubated with or without various concentrations of compound
11, WK-X-34 or VRP (1.0, 2.5, 5 µM) for another 90 min. Afterwards the cells were
washed twice in ice-cold PBS. The mean fluorescence intensity of retained
intracellular Rh123 was estimated by BD FACSCalibur flow cytometer.
Acknowledgments
This work was supported by the National Science and Technology Major Project
of the Ministry of Science and Technology of China (NO. 2009ZX09102-033) and
National Natural Science Foundation of China (NO. 81173088).
Notes and references
a
Author information. Center of Drug Discovery, State Key Laboratory of Natural

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Medicines,, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P. R.
b
China. Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, P. R. China.
Corresponding authors.*Wenglong Huang: phone: +86-25-83271302; fax:
+86-25-83271302; E-mail: ydhuangwenlong@126.com; *Hai Qian: phone:
+86-25-83271302; fax: +86-25-83271320; E-mail: qianhai24@163.com.
Conflict of interest. All authors report no conflict of interest.
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55, 1999-2014.
Figure caption
Fig. 1 Structures of Verapamil, VX-710, Zosuquidar and Tariquidar
Fig. 2 Structure of WK-X-34 and design of the target compounds
Scheme 1 Synthesis of the target compounds. Reagents and conditions: (i)
3-bromoprop-1-yne, K2CO3, acetone, reflux, 3 h; (ii) Fe/NH4Cl, 80% EtOH, reflux, 2.5 h;
(iii) heterocyclic aromatic acyl chlorides, TEA/DCM, r.t., 24 h; or heterocyclic aromatic
formic acids, EDC·HCl/DMAP/DCM, r.t., 48 h; (iv) 1-(2-bromoethyl)-4-nitrobenzene,
K2CO3, acetonitrile, reflux, 17 h; (v) H2/Pd/C, DCM/EtOH, r.t., 24 h; (vi) NaNO2, 50%
AcOH, 0-5 °C, 30 min; NaN3, 0-5 °C, 50 min; (vii) c1-c12, ascorbate sodium, CuSO4,
75%CH3OH, 24-48 h.

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Table 1 Cytotoxicity of the target compounds against K562 and K562/A02 cell lines.
The IC50s for the target compounds were determined by MTT method. Each
experiment was carried out three times.
Table 2 ADM-resistance reversal activity of the target compounds 1-12 at 5µM
concentration in K562/A02 cells. The IC50 value was determined after exposure to a
series of ADM concentration with different target compounds at 5 µM using
K562/A02 cells. Reversal fold (RF) refers to fold-change in drug sensitivity. RF= (IC50
without modulator)/ (IC50 with 5 µM modulator).0.1% DMSO was added as solvent
control for testing the P-gp modulating activity.
Table 3 Sensitization of compound 11 on reversing MDR towards K562/A02 cells at
different concentrations. Data were analyzed with GraphPad Prism 5.0 software and
are the mean ± SD for 3 independent tests. The IC50 of ADM was 82.21 ± 3.7 µM.
Reversal fold (RF), RF= (IC50 without modulator)/ (IC50 with modulator).
Fig. 3 EC50 for the compound 11 in K562/A02 cells. Various concentrations (0.10,
0.25.0.50, 1.00, 2.50, 5.00, 10.00, 20.00 µM) of compound 11 and ADM were added
to 96-well plates and the cells were incubated for 48 h. The absorption values were
measured with a Microplate Reader at 490 nm. Data were analyzed with GraphPad
Prism 5.0 software and are the mean ± SD for 3 independent tests.
Table 4 Duration of MDR reversal in K562/A02 cells after incubation and washout of
P-gp inhibitor. Numbers in parentheses, Reversal fold (RF), RF= (IC50without
modulator)/ (IC50with modulator).Each experiment was carried out two to three
times, and the values were presented as the mean ± standard error of mean. Nd: not

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determined.
Fig. 4 Effect of compound 11 on intracellular ADM accumulation in K562/A02 cells.
0.1% DMSO was used as negative control. VRP and WK-X-34 were chosen as positive
controls. The mean fluorescence intensity of retained intracellular ADM was
estimated by spectrofluorometry. Data were expressed as means ± S.D. of 3
independent experiments. *P<0.05, **P<0.01 vs. untreated K562/A02 cells.
Fig. 5. Inhibitory effect of compound 11 on the efflux of Rh123. K562 or K562/A02
cells were incubated with 5 µM Rh123 for 60 min. Then various concentrations of
compound 11, WK-X-34 or VRP (1.0, 2.5, 5µM) was added and the cells were
incubated for a further 90 min. Afterwards the cells were washed 3 times in ice-cold
PBS. The mean fluorescence intensity of retained intracellular Rh123 was estimated
by flow cytometry. Data were expressed as means ± S.D. of 3 independent
experiments. *P<0.05, **P<0.01 vs. untreated K562/A02 cells.

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176x124mm (300 x 300 DPI)