Discovery to solve multidrug resistance: Design, synthesis, and biological evaluation of novel agents

Article abstract of DOI:10.1002/ardp.201900127

Chemotherapy remains a pillar in the treatment and management of various cancers. However, multidrug resistance (MDR) becomes a severe problem after long-term administration of chemotherapy drugs. Overexpression of P-glycoprotein (P-gp) is a significant cause for tumor MDR. Therefore, P-gp inhibition is considered as an effective strategy to reverse MDR. A third-generation P-gp inhibitor tariquidar was selected as a lead compound, and a new series of triazol-N-ethyl tetrahydroisoquinoline based compounds were designed as novel P-gp inhibitors and synthesized through click chemistry. These compounds presented higher reversal activities than the positive-control verapamil (VRP). Among 18 compounds, compound 11 without cytotoxicity reversed MDR in a dose-dependent manner, with a persistent longer chemosensitizing effect and reversibility compared to others. Mechanism studies discovered that compound 11 could escalate the intracellular accumulation of rhodamine-123 and doxorubicin in K562/A02 cells as well as inhibit their efflux from cells. The results obtained suggest that compound 11 is more potent than VRP administered under the same conditions; it may be a potent and safe candidate for P-gp modulation for further development.

Full text of DOI:10.1002/ardp.201900127

Received: 23 April 2019
Revised: 11 July 2019
Accepted: 17 July 2019
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DOI: 10.1002/ardp.201900127
F U L L P A P E R
Discovery to solve multidrug resistance: Design, synthesis,
and biological evaluation of novel agents
Qianqian Qiu1*
Shaoyang Zhou1
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Wei Shi2*
Mutta Kairuki2
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Shiyuan Zhao1*
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Yan Zhu1
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Zhengquan Ding1
Hai Qian2,3
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Wenlong Huang2,3
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1School of Pharmacy, Jiangsu Provincial Key
Laboratory of Coastal Wetland Bioresources
and Environmental Protection, Yancheng
Teachers’ University, Yancheng, China
Abstract
Chemotherapy remains a pillar in the treatment and management of various cancers.
However, multidrug resistance (MDR) becomes a severe problem after long‐term
administration of chemotherapy drugs. Overexpression of P‐glycoprotein (P‐gp) is a
significant cause for tumor MDR. Therefore, P‐gp inhibition is considered as an
effective strategy to reverse MDR. A third‐generation P‐gp inhibitor tariquidar was
selected as a lead compound, and a new series of triazol‐N‐ethyl tetrahydroisoquino-
line based compounds were designed as novel P‐gp inhibitors and synthesized
through click chemistry. These compounds presented higher reversal activities than
the positive‐control verapamil (VRP). Among 18 compounds, compound 11 without
cytotoxicity reversed MDR in a dose‐dependent manner, with a persistent longer
chemosensitizing effect and reversibility compared to others. Mechanism studies
discovered that compound 11 could escalate the intracellular accumulation of
rhodamine‐123 and doxorubicin in K562/A02 cells as well as inhibit their efflux from
cells. The results obtained suggest that compound 11 is more potent than VRP
administered under the same conditions; it may be a potent and safe candidate for
P‐gp modulation for further development.
2State Key Laboratory of Natural Medicines,
Center of Drug Discovery, China
Pharmaceutical University, Nanjing, China
3Jiangsu Key Laboratory of Drug Discovery
for Metabolic Disease, China Pharmaceutical
University, Nanjing, China
Correspondence
Prof. Wenlong Huang and Dr. Hai Qian, State
Key Laboratory of Natural Medicines, Centre
of Drug Discovery, China Pharmaceutical
University, 24 Tongjiaxiang, 210009 Nanjing,
China.
Email: ydhuangwenlong@126.com (W. H.) and
qianhai24@163.com (H. Q.)
Funding information
The Foundation of Jiangsu Provincial Key
Laboratory of Coastal Wetland Bioresources
and Environmental Protection, Grant/Award
Number: JKLBS2017011; National Natural
Science Foundation of China, Grant/Award
Numbers: 81872734, 81803353, 81872733
K E Y W O R D S
click chemistry, multidrug resistance, P‐glycoprotein inhibitor, reversal activity
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1
INTRODUCTION
transport proteins known as ATP‐binding cassette (ABC) transpor-
ters, which escalate efflux of chemotherapeutic drugs from
Cancer is a severe disease without a highly effective cure as of now.
It usually arises from a single stem cell with the ability for self‐
renewal using intrinsic cellular properties and environmental factors.
The success of anticancer chemotherapy is always hindered by drug
resistance, which acts as a defensive mechanism that the tumor cells
cultivate against chemotherapeutic drugs.[1] Multidrug resistance
(MDR) is a type of acquired drug resistance to multiple classes of
structurally and mechanistically unrelated anticancer drugs.[2] The
resistance occurs due to overexpression of membrane‐associated
tumor cells.[3] ABC transporters mainly consist of breast cancer
(BCRP/ABCG2), multidrug resistance protein (MRP/ABCC1)
1
and P‐glycoprotein (P‐gp/ABCB1). Among them, P‐gp is the most
studied of the ABC transporters and plays an important role in
drug efflux.[4,5]
P‐gp is quantified in various crucial tissues and blood–tissue
barriers, where it plays important physiological roles such as
extrusion of exogenous and endogenous toxic agents that enter the
body and direct of the secretion of lipophilic molecules.[6] However,
P‐gp is overexpressed in tumor cells as a result of regulation of the
human gene expression of MDR1 that causes an accelerated efflux of
the chemotherapeutic drugs, inducing classical multidrug resistance
*Qianqian Qiu, Wei Shi, and Shiyuan Zhao contributed equally as first authors.
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Arch. Pharm. Chem. Life Sci. 2019;e1900127.
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(MDR).[7] P‐gp uses ATP hydrolysis for energy to pump drugs out of
the cell and has various substrate specificities.[8] Coadministration of
a P‐gp inhibitor together with a primary chemotherapeutic agent is
suggested as an effective approach to overcome MDR.[9] Over many
decades, three generations of P‐gp inhibitors have been developed:
verapamil (first generation),[10] dexverapamil (second generation),[11]
and tariquidar (third generation[12]; Figure 1). However, there is no P‐
gp inhibitor that has been approved for clinical use due to their poor
potency, unsatisfactory toxicity, and low selectivity properties.[13]
For designing new MDR reversal agents, we selected tariquidar
as the lead compound and designed the structure of triazole as
bioisosteres to replace benzene, the amido bond was modified by the
secondary amine. We linked various chemical structures with
aromatic amides through the click chemistry method.[14] The 1,2,3‐
tariquidar show weak cytotoxicity to K562 cells. The IC50 value of
DOX in resistant K562/A02 cells was almost 46‐fold that in K562
cells.
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2.2.2
Effects of the target compounds on reversing
DOX resistance in K562/A02 cells
According to the cytotoxicity assay results, the tested compound
shows no cytotoxicity to K562 and K562/A02 cells at a concentra-
tion of 5 µM. The properties of the synthesized compounds on
reversing DOX resistance towards human erythroleukemia adriamy-
cin‐resistant K562/A02 cells (P‐gp overexpression) were tested by
MTT assay at such a concentration. The results are shown in Table 3.
Among them, compounds 6, 10, 11, 12 show obvious reversal activity
towards K562/A02 cells. And compound 11 is much better than the
positive‐control VRP and similar to tariquidar. The reversal fold (RF)
value of compound 11 reached 18.7.
triazole ring,
a hydrophobic aromatic group, associating with
biological targets through hydrogen bonding and dipole interactions
was introduced to the designed compounds (Figure 2). Click
chemistry, commonly copper(I)‐catalyzed 1,2,3‐triazole formation
from azides and terminal acetylenes, has been widely applied in
drug discovery.[15] Applying the procedure illustrated, we synthe-
sized and biologically evaluated compounds 1–18 as P‐gp‐mediated
MDR reversal agents.[16]
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2.2.3
Chemosensitizing effect of compound 11
Furthermore, we investigated compound 11 for its reversal potency
and dose–response effects using different concentrations such as 20,
10, 5, 2.5, 1.25, 0.625, 0.31, 0.156, and 0.078 µM towards K562/A02
cells by MTT assay taking VRP as a positive control. As shown in
Table 4, compound 11 displayed a dose‐dependent reversal activity.
Moreover, the EC50 value of compound 11 was 590.6 3.7 nM
(Figure 3), which was premeditated using GraphPad Prism 5.0
software from the dosage–response curves. The outcomes suggested
that compound 11 had significant potential to develop the sensitivity
of P‐gp overexpressing cells to anticancer drug substrates in a dose‐
dependent manner.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthetic routes of the target compounds 1–18 are delineated in
Scheme 1. The detailed synthesis procedures are shown in Section 4
(Table 1Aa and 1Ab).
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2.2
Biological evaluation
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Duration of MDR reversal effect of
compound 11 for DOX in K562/A02 cells
2.2.1
cells
Cytotoxicity assay on K562 and K562/A02
2.2.4
The cytotoxicity of the synthesized compounds towards K562 and
doxorubicin (DOX)‐resistant K562/A02 cells were determined by 3‐
(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT)
assay.[17,18] As shown in Table 2, most of the tested compounds
showed no cytotoxicity to K562 and K562/A02 cells, their IC50
values were greater than 40 µM. Compounds 6, 8, 13, VRP, and
The desired P‐gp inhibitor should possess a lasting effect over a
certain period. The duration of the MDR reversal effect was
accessed to verify the reversal activity of compound 11. Data in
Table 5 shows that the MDR reversal effect of positive‐control
VRP did not last more than 6 hr. However, compound 11 presented
reversal activity even after its removal from the medium for 12 hr
FIGURE 1 Structures of the known
P‐glycoprotein inhibitors

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FIGURE 2 Design of the target
compounds
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and the IC50 was 8.65 0.64 µM (RF = 2.96). These data illustrate
that compound 11 displayed a potent MDR reversing effect and
persevered for a longer time compared with the positive‐control
VRP.
2.2.6
Inhibitory effect of compound 11 on the P‐gp
efflux function
For further verification of our assumption, the inhibitory effect of
compound 11 on the efflux function of P‐gp was analyzed by spotting the
reserved intracellular rhodamine‐123 (Rh123), a fluorescent substrate of
P‐gp. As indicated in Figure 5, the fluorescence intensity of Rh123 in P‐gp
overexpressed K562/A02 cells decreased significantly over time. When
5 µM VRP or compound 11 was added in K562/A02 cells, the degree of
fluorescence intensity increased, which denotes VRP or compound 11
helps inhibit efflux of Rh123 out of cells. At the same concentration, the
effect of compound 11 was better than that of VRP.
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2.2.5
Effect of compound 11 on DOX
accumulation in K562/A02 cells
To confirm our results, we assessed the accumulation of doxorubicin
as a fluorescence substrate of P‐gp in K562/A02 cells by fluores-
cence spectrophotometry.[19,20] As detailed in Figure 4, DOX
accumulation in DOX‐sensitive K562 cells is far more than in DOX‐
resistant K562/A02 cells. Compared with blank control, DOX
accumulation increased in varying degrees after treatment with
VRP or compound 11. And even at the low concentration of 0.5 µM,
compound 11 still increased DOX accumulation in K562/A02 cells.
These data illustrated that compound 11 could inhibit the function of
P‐gp, causing DOX accumulation.
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2.3
Structure–activity relationships
The study of structure–activity relationship suggests that various
substituents in R of compounds 1–18 could affect MDR reversal
FIGURE 3 EC50 value of compound 11 in reversing doxorubicin
(DOX)‐resistance in K562/A02 cells. The percentage of DOX IC50
was charted with a log concentration of 11, which is equal to (DOX
IC50 in each modulator concentration/DOX IC50 without
FIGURE 4 The effect of compound 11 on intracellular DOX
accumulation in K562/A02 cells. The results are presented as the
mean SD (standard deviation) for three independent experiments;
**p < 0.01, *p < 0.05 relative to the negative control (K562/A02).
DOX, doxorubicin; SD, standard deviation; VRP, verapamil
modulator) × 100%. Thus, EC50 can be noted for the modulator
concentration, which can diminish the DOX IC50 by 50%

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3
CONCLUSION
In this study, a new series of novel P‐gp inhibitors has been designed,
synthesized and evaluated for biological activity. Results demonstrated
that a few of them possess a certain MDR reversal activity. In particular,
among them, compound 11 shows a potent effect compared with the
positive control. The EC50 value of compound 11 is 590.6 3.7 nM.
Compound 11 shows no obvious cytotoxicity towards K562 and K562/
A02 cells. The MDR reversal effect of compound 11 could last over a
certain period, longer than that of VRP. Compound 11 shows
remarkable potency in increased accumulation of doxorubicin in
K562/A02 cells. And it also obviously decreases P‐gp‐mediated efflux
of Rh123 out of cells. In conclusion, compound 11 should be appropriate
as a potential P‐gp modulator for further study.
FIGURE 5 Effect of compound 11 on efflux of Rh123 from K562/
A02. Each data point is presented as mean SD for three
independent experiments. MFI, mean fluorescence intensity; Rh123,
rhodamine‐123; SD, standard deviation; VRP, verapamil
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4
EXPERIMENTAL
Chemistry
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4.1
activities on K562/A02 cells. The electron‐donating group in R can be
of great importance for the activity. The preferred compound 11
possesses three methoxy groups, which contribute a lot to its good
potency. Comparing compounds 1–13 with compounds 14–18, the
activity of the latter group is obviously worse than the former, which
demonstrates 3,4‐dimethoxy on the benzene ring can increase the
reversal activity because more interaction with P‐gp has occurred.
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4.1.1
General
The structures of the synthesized compounds were characterized by
1H NMR and 13C NMR spectroscopy (Bruker ACF‐300Q, 300 MHz),
with Me4Si as an internal standard and dimethyl sulfoxide (DMSO‐
d6) as a solvent. Melting points were taken on an RY‐1 melting‐point
apparatus. Purification by column chromatography was carried out
SCHEME 1 Synthesis of the target compounds. Reagents and conditions: (i) 3‐bromoprop‐1‐yne, K2CO3, acetonitrile, 80℃, reflux, 6 hr;
(ii) LiOH, 80% MeOH, 64℃, 1 hr; (iii) RNH2, EDCI/HOBt, DCM r.t., 3–5 hr; (iv) NaN3, water, 80℃, 24 hr; (v) TEA/DCM, TsCl, r.t., 24 hr; (vi)
paraformaldehyde, EtOH, HCl, 78℃, reflux, 5 hr; (vii) TEA, acetonitrile, 60℃, 24 hr; (viii) 1.0 eq. g, ascorbate sodium, CuSO4, 75% MeOH,
24–48 hr; (ix) 3‐bromoprop‐1‐yne, K2CO3, acetonitrile, 80℃, reflux, 6 hr; (x) RCOOH, EDCI/HOBt, DCM r.t., 3–5 hr; (xi) 1.0 eq. g, ascorbate
sodium, CuSO4, 75% CH3OH, 24–48 hr. r.t., room temperature

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TABLE 1A Structures of targeted compounds 1–13
TABLE 2 Cytotoxicity of compounds towards K562 and K562/
A02 cell lines
Cytotoxicity IC50 (µM)
Compounds
K562
K562/A02
>100
1
48.77 1.21
>100
2
>100
3
45.22 1.33
>100
>100
Compound
1
R
Compound
8
R
4
>100
5
>100
>100
6
32.02 1.25
>100
>100
7
>100
2
9
8
40.31 3.05
>100
>100
9
>100
10
>100
>100
11
>100
>100
3
4
10
11
12
>100
>100
13
26.10 1.53
>100
>100
14
>100
15
>100
>100
16
>100
>100
17
>100
>100
5
12
13
18
>100
>100
VRP
Tariquidar
DOX
32.24 2.89
20.78 1.44
0.53 0.06
35.13 2.05
33.11 3.66
24.31 0.86
6
7
Abbreviations: DOX, doxorubicin; RF, reversal fold; VRP, verapamil.
over silica gel (100–200 or 200–300 mesh); the reactions were
monitored by thin‐layer chromatography on GF/UV254 plates and
were visualized using UV light at 254 or 365 nm. The electrospray
ionization mass spectrometry (ESI–MS) data were recorded on
Waters ACQUITY UPLC systems with mass (Waters, Milford, MA).
All chemical reagents were obtained from commercial sources and
used without purification unless otherwise indicated.
TABLE 1B Structures of targeted compounds 14–18
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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4.1.2
General procedure for preparation of
compounds 1–18
The synthetic routes to target compounds 1–18 are shown in Scheme
1. For compounds 1–13, first, methyl 2‐amino‐4,5‐dimethoxybenzo-
Compound
14
R
Compound
17
R
ate and 3‐bromo‐prop‐1‐yne were refluxed in
a mixture of
acetonitrile and potassium carbonate for 6 hr 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, followed by
1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDCI) and 1‐hydro-
xybenzotriazole (HOBT) to give compounds c1–c13. 2‐Bromoethanol
and sodium azide in water were stirred at 80°C for 24 hr, and then
15
16
18

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TABLE 3 DOX resistance reversal activity of the target compounds 1–18 at 5 µM concentration in K562/A02 cells
Compound (5.0 μM)
IC50/DOX (μM)
26.13 0.56
19.32 0.18
18.85 0.65
39.85 0.84
14.82 0.45
4.23 0.39
22.92 0.48
7.51 0.36
6.22 0.33
5.12 0.25
1.30 0.35
RF
0.93
Compound (5.0 μM)
IC50/DOX (μM)
3.72 0.24
16.26 0.25
15.63 0.14
10.35 0.39
9.25 0.26
18.20 0.33
9.33 0.86
5.23 0.26
1.42 0.15
24.31 0.86
RF
1
12
6.53
1.50
1.56
2.35
2.63
1.34
2.61
4.66
17.12
1
2
1.26
1.29
0.61
1.64
5.60
1.06
3.24
3.91
4.75
13
3
14
4
15
5
16
6
17
7
18
8
VRP
Tariquidar
Control
9
10
11
18.7
Abbreviations: DOX, doxorubicin; RF, reversal fold; VRP, verapamil.
3 hr, the mixture was adjusted to pH = 2 with concentrated
hydrochloric acid, then refluxed for 4 hr, and then cooled down to
afford compound f. To a solution of compound e in dry acetonitrile
and triethylamine, compound f was added for 24 h‐reflux. Column
chromatography on a silica gel column (EtOAc/PE 3:2 to EtOAc/
MeOH 16:1) was used to give compound g.[16,21,22] For compounds
14–18, o‐phenylenediamine and 3‐bromo‐prop‐1‐yne were refluxed
in a mixture of acetonitrile and potassium carbonate for 6 hr to
afford compound h, which was then treated with the substituted
benzoic acid (DIEA) in dry dichloromethane, followed by EDCI and
HOBT to give compounds i14–i18. To the solution of c1–13 or
i14–18 (1 mmol) and intermediates g (1 mmol) in 75% methanol
(40 ml), ascorbate sodium (30 mg), and CuSO4 (10 mg) were added
for stirring at room temperature for 24–48 hr. The crude product
was purified by chromatography on silica gel utilizing a mixture of
chloroform and acetone (80:1, v/v) as eluent to furnish the desired
target compounds 1–18.
TABLE 4 Sensitization of compound 11 on reversing MDR toward
K562/A02 cells at different concentrations
Compound
IC50 of DOX (µM)
24.31 0.86
5.23 0.26
RF
None
1
VRP, 5 µM
4.66
Compound 11, 20 µM
Compound 11, 10 µM
Compound 11, 5 µM
Compound 11, 2.5 µM
Compound 11, 1.25 µM
Compound 11, 0.625 µM
Compound 11, 0.31 µM
Compound 11, 0.156 µM
Compound 11, 0.078 µM
0.73 0.29
33.30
25.06
17.12
5.56
3.12
1.89
1.54
1.30
1.14
0.97 0.14
1.42 0.15
4.37 0.34
7.78 0.74
12.88 0.31
15.80 0.29
18.71 0.53
21.39 0.28
Abbreviations: DOX, doxorubicin; MDR, multidrug resistance; RF, reversal
fold.
extracted with ethyl acetate. The solvent was removed under
vacuum to get compound d. Compound d was reacted with 4‐
toluenesulfonylchloride and triethylamine in dry dichloromethane to
get compound e. 3,4‐Dimethoxyphenethylamine, paraformaldehyde,
and absolute ethanol were firstly stirred at room temperature for
N‐Benzyl‐2‐(((1‐(2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐
1H‐1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxybenzamide (1)
Yield: 75.3%; brown powder; mp: 145–147℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 8.70 (s, 1H), 8.42 (s, 1H), 8.00 (s, 1H), 7.41–7.17 (m,
6H), 6.60 (d, J = 12.3 Hz, 2H), 6.41 (s, 1H), 4.54 (s, 2H), 4.40 (s, 4H),
3.71 (d, J = 13.7 Hz, 12H), 3.49 (s, 2H), 2.88 (s, 2H), 2.64 (s, 4H). 13C
NMR (125 MHz, DMSO‐d6): δ = 167.25, 151.82, 147.22, 147.18,
145.32, 142.51, 141.04, 138.30, 128.75, 127.56, 127.28, 127.17,
126.31, 121.32, 114.18, 112.52, 111.32, 111.01, 101.01, 56.35,
56.17, 56.08, 56.00, 55.99, 54.58, 50.81, 48.90, 44.82, 36.64, and
28.77. ESI–MS m/z: 587.1 [M+H]+. Anal. calcd. for C32H38N6O5: C,
65.51; H, 6.53; N, 14.32; Found: C, 65.54; H, 6.51; N, 14.34.
TABLE 5 Duration of MDR reversal in K562/A02 cell after
incubation and washout of VRP or compound 11
IC50/DOX (µM) (RF)
Treatment
schedule
Compound 11
(5 µM)
Control
VRP (5 µM)
No wash
25.59 1.87 3.55 0.61 (7.21) 1.31 0.12 (19.53)
(1.00)
Wash 0 hr
Wash 6 hr
Wash 12 hr
ndb
12.54 1.93 (2.04) 2.35 0.17 (10.89)
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐N‐(3,4‐dimethoxyphenethyl)‐4,5‐di-
nd
nd
nd
5.31 0.34 (4.82)
8.65 0.64 (2.96)
nd
methoxybenzamide ( )
2
Yield: 72.8%; brown powder; mp: 142–143℃. 1H NMR (300 MHz,
Abbreviations: DOX, doxorubicin; MDR, multidrug resistance; RF, reversal
fold; VRP, verapamil.
DMSO‐d6): δ = 8.33 (t, J = 5.3 Hz, 1H), 8.20 (d, J = 4.5 Hz, 1H), 8.02 (s,

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1H), 7.15 (s, 1H), 6.91–6.79 (m, 2H), 6.74 (d, J = 8.1 Hz, 1H), 6.60 (d,
J = 11.3 Hz, 2H), 6.40 (s, 1H), 4.55 (t, J = 6.0 Hz, 2H), 4.39 (d,
J = 5.3 Hz, 2H), 3.86–3.65 (m, 19H), 3.54 (s, 2H), 2.89 (t, J = 6.0 Hz,
2H), 2.74 (t, J = 7.3 Hz, 2H), 2.66 (s, 3H). 13C NMR (75 MHz, DMSO‐
d6): δ = 167.57, 152.12, 149.20, 147.89, 147.19, 147.14, 145.42,
142.81, 141.02, 132.23, 127.27, 126.55, 121.33, 121.31, 114.33,
112.47, 112.29, 112.20, 111.54, 111.29, 101.04, 56.37, 56.16, 56.01,
55.99, 55.97, 55.91, 54.59, 50.65, 48.86, 40.67, 36.64, 34.74, and
28.78. ESI–MS m/z: 661.3 [M+H]+. Anal. calcd. for C35H44N6O7: C,
63.62; H, 6.71; N, 12.72; Found: C, 63.54; H, 6.65; N, 12.70.
50.81, 48.90, 36.64, and 28.77. ESI–MS m/z: 573.6 [M+H]+. Anal.
calcd. for C31H36N6O5: C, 65.02; H, 6.34; N, 14.68; Found: C, 65.09;
H, 6.28; N, 14.65.
N‐(4‐Bromophenyl)‐2‐(((1‐(2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐
2(1H)‐yl)ethyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy-
benzamide (6)
Yield: 70.5%; brown powder; mp: 149–151℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.92 (s, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.57 (2d, 7.7 Hz,
4H), 7.34 (s, 1H), 6.59 (d, J = 9.0 Hz, 2H), 6.46 (s, 1H), 4.49 (m, 4H),
3.96–3.56 (m, 12H), 3.52 (s, 2H), 2.88 (s, 2H), 2.64 (s, 4H). 13C NMR
(75 MHz, DMSO‐d6): δ = 165.11, 152.04, 147.22, 147.18, 145.27,
143.20, 141.04, 137.29, 131.50, 127.17, 126.60, 123.62, 121.32,
118.15, 114.80, 112.50, 111.66, 111.00, 101.20, 56.34, 56.16, 56.08,
56.00, 55.99, 54.58, 50.81, 48.86, 36.64, and 28.76. ESI–MS m/z:
652.4 [M+H]+. Anal. calcd. for C31H35BrN6O5: C, 57.15; H, 5.41; N,
12.90; Found: C, 57.19; H, 5.38; N, 12.84.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐(4‐methoxyphe-
nyl)benzamide (3)
Yield: 64.5%; brown powder; mp: 153–156℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.70 (s, 1H), 8.09 (t, J = 5.4 Hz, 1H), 8.02 (s, 1H), 7.52 (d,
J = 8.9 Hz, 2H), 7.33 (s, 1H), 6.90 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 10.3 Hz,
2H), 6.44 (s, 1H), 4.54 (t, J = 6.0 Hz, 2H), 4.42 (d, J = 5.3 Hz, 2H), 3.75 (d,
J = 7.0 Hz, 9H), 3.67 (s, 6H), 3.52 (s, 2 H), 2.88 (t, J = 6.0 Hz, 2H), 2.64 (s,
4H). 13C NMR (75 MHz, DMSO‐d6): δ = 165.11, 156.59, 152.02,
147.22, 147.18, 145.27, 143.19, 141.01, 133.57, 127.17, 126.60,
122.97, 121.32, 114.96, 114.34, 112.52, 111.63, 111.01, 101.07,
56.35, 56.16, 56.00, 55.99, 55.35, 54.58, 50.65, 48.86, 36.64, and
28.76. ESI–MS m/z: 663.3 [M+H]+. Anal. calcd. for C32H38N6O6: C,
63.77; H, 6.36; N, 13.94; Found: C, 63.74; H, 6.32; N, 13.89.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐phenethylbenza-
mide (7)
Yield: 62.3%; brown powder; mp: 156–157℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 8.32 (d, J = 5.0 Hz, 1H), 8.23 (s, 1H), 8.02 (s, 1H), 7.24
(m, 6H), 6.60 (d, J = 10.8 Hz, 2H), 6.40 (s, 1H), 4.55 (t, J = 5.5 Hz, 2H),
4.39 (d, J = 5.0 Hz, 2H), 3.71 (d, J = 15.4 Hz, 12H), 3.54 (s, 2H),
3.45–3.37 (m, 2H), 2.85 (dt, J = 14.9, 6.4 Hz, 4H), 2.66 (s, 4H). 13C
NMR (75 MHz, DMSO‐d6): δ = 167.57, 151.82, 147.22, 147.18,
145.32, 142.51, 141.01, 138.74, 128.94, 128.76, 127.17, 126.79,
126.60, 121.32, 114.45, 112.52, 111.32, 111.01, 101.01, 56.35,
56.16, 56.00, 55.99, 54.58, 50.65, 48.90, 40.82, 36.64, 34.78, and
28.76. ESI–MS m/z: 601.5 [M+H]+. Anal. calcd. for C33H40N6O5: C,
65.98; H, 6.71; N, 13.99; Found: C, 65.94; H, 6.74; N, 13.91.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐N‐isobutyl‐4,5‐dimethoxybenza-
mide (4)
Yield: 58.4%; brown powder; mp: 156–158℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 8.31 (t, J = 5.3 Hz, 1H), 8.14 (t, J = 5.5 Hz, 1H), 8.01 (s,
1H), 7.21 (s, 1H), 6.60 (d, J = 13.3 Hz, 2H), 6.39 (s, 1H), 4.55 (t,
J = 6.0 Hz, 2H), 4.38 (d, J = 5.2 Hz, 2H), 3.71 (d, J = 11.0 Hz, 12H), 3.53
(s, 2H), 3.00 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 6.0 Hz, 2H), 2.65 (s, 2H),
1.81 (dt, J = 13.3, 6.6 Hz, 1H), 0.87 (d, J = 6.6 Hz, 8H). 13C NMR
(75 MHz, DMSO‐d6): δ = 167.81, 151.82, 147.23, 147.18, 145.39,
142.57, 141.04, 127.25, 126.35, 121.33, 114.18, 112.52, 111.28,
111.02, 101.02, 56.38, 56.18, 56.08, 56.06, 56.04, 54.58, 50.86,
48.90, 47.69, 36.64, 28.79, 28.23, and 20.12. ESI–MS m/z: 553.5
[M+H]+. Anal. calcd. for C29H40N6O5: C, 63.02; H, 7.30; N, 15.21;
Found: C, 63.10; H, 7.35; N, 15.17.
N‐(4‐Chlorophenyl)‐2‐(((1‐(2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐
2(1H)‐yl)ethyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy-
benzamide (8)
Yield: 71.2%; brown powder; mp: 142–144℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.94 (s, 1H), 8.19–8.02 (m, 2H), 7.85 (s, 1H), 7.60 (d,
J = 8.2 Hz, 1H), 7.41–7.32 (m, 2H), 7.12 (d, J = 7.9 Hz, 1H), 6.59 (d,
J = 8.6 Hz, 2H), 6.47 (s, 1H), 4.55 (t, J = 5.8 Hz, 2H), 4.45 (d, J = 5.1 Hz,
2H), 3.82–3.64 (m, 12H), 3.53 (s, 2H), 2.89 (t, J = 5.8 Hz, 2H), 2.65 (s,
4H). 13C NMR (75 MHz, DMSO‐d6): δ = 165.11, 152.02, 147.22,
147.18, 145.27, 143.19, 141.04, 137.07, 128.62, 127.84, 127.17,
126.60, 122.50, 121.32, 114.80, 112.52, 111.63, 111.01, 101.07,
56.35, 56.16, 56.08, 56.00, 55.99, 54.58, 50.81, 48.86, 36.64, and
28.76. ESI–MS m/z: 607.1 [M+H]+. Anal. calcd. for C31H35ClN6O5: C,
61.33; H, 5.81; N, 13.84; Found: C, 61.38; H, 5.74; Cl, 5.81; N, 13.79.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐phenylbenzamide
( )
5
Yield: 72.5%; brown powder; mp: 142–144℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.81 (s, 1H), 8.05 (d, J = 8.9 Hz, 2H), 7.63 (d, J = 7.8 Hz,
2H), 7.40–7.23 (m, 3H), 7.07 (t, J = 7.2 Hz, 1H), 6.59 (d, J = 9.8 Hz, 2H),
6.46 (s, 1H), 4.48 (2d, J = 5.3 Hz, 4H), 3.82–3.59 (m, 12H), 3.52 (s, 2H),
2.87 (d, J = 5.6 Hz, 2H), 2.64 (s, 4H). 13C NMR (75 MHz, DMSO‐d6):
δ = 165.11, 152.02, 147.22, 147.18, 145.27, 143.19, 141.04, 138.68,
128.80, 127.27, 126.35, 124.13, 121.33, 121.11, 114.80, 112.52,
111.63, 111.02, 101.07, 56.37, 56.17, 56.08, 56.03, 56.02, 54.58,
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐N‐(4‐fluorophenyl)‐4,5‐dimethoxy-
benzamide (9)
Yield: 59.3%; brown powder; mp: 150–151℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.86 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.64 (dd, J = 8.6,

8 of 10
QIU ET AL.
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5.2 Hz, 2H), 7.34 (s, 1H), 7.16 (t, J = 8.8 Hz, 2H), 6.60 (d, J = 10.3 Hz,
2H), 6.46 (s, 1H), 4.55 (t, J = 5.7 Hz, 2H), 4.43 (d, J = 5.0 Hz, 2H), 3.73
(dd, J = 16.8, 9.4 Hz, 12H), 3.53 (s, 2H), 2.89 (t, J = 5.8 Hz, 2H), 2.65 (s,
4H). 13C NMR (75 MHz, DMSO‐d6): δ = 165.11, 160.53, 158.51,
152.02, 147.22, 147.18, 145.27, 143.19, 141.04, 135.74, 135.71,
127.17, 126.31, 123.07, 123.01, 121.32, 115.75, 115.59, 115.00,
112.52, 111.63, 111.01, 101.07, 56.35, 56.17, 56.08, 56.00, 55.99,
54.58, 50.81, 48.90, 36.64, and 28.76. ESI–MS m/z: 591.5 [M+H]+.
Anal. calcd. for C31H35FN6O5: C, 63.04; H, 5.97; N, 14.23; Found: C,
63.08; H, 5.94; N, 14.27.
m/z: 629.6 [M+H]+. Anal. calcd. for C35H44N6O5: C, 66.86; H, 7.05; N,
13.37; Found: C, 66.81; H, 7.10; N, 13.34.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐(2‐methoxyphe-
nyl)benzamide (13)
Yield: 69.3%; brown powder; mp: 146–148℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.25 (s, 1H), 8.19–7.92 (m, 2H), 7.57 (d, J = 6.3 Hz, 1H),
7.35 (d, J = 5.4 Hz, 1H), 7.19–6.86 (m, 3H), 6.68–6.52 (m, 2H), 6.45 (d,
J = 5.4 Hz, 1H), 4.64–4.36 (m, 4H), 3.98–3.63 (m, 15H), 3.52 (s, 2H),
2.86 (d, J = 5.2 Hz, 2H), 2.64 (s, 4H). 13C NMR (75 MHz, DMSO‐d6):
δ = 165.33, 152.02, 150.67, 147.22, 147.18, 145.14, 143.19, 141.01,
128.26, 127.17, 126.60, 125.56, 123.00, 121.32, 121.31, 114.98,
114.37, 112.52, 111.61, 111.01, 101.00, 56.35, 56.16, 56.00, 55.99,
55.69, 54.58, 50.65, 48.86, 36.64, and 28.76. ESI–MS m/z: 603.5
[M+H]+. Anal. calcd. for C32H38N6O6: C, 63.77; H, 6.36; N, 13.94;
Found: C, 63.75; H, 6.34; N, 13.98.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐(o‐tolyl)benzamide
(10)
Yield: 68.1%; brown powder; mp: 147–149℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.77 (s, 1H), 8.06 (d, J = 18.1 Hz, 2H), 7.55 (d,
J = 8.4 Hz, 2H), 7.33 (d, J = 4.8 Hz, 3H), 6.69–6.53 (m, 2H), 6.45 (s,
1H), 4.62–4.34 (m, 4H), 3.84–3.59 (m, 12H), 3.52 (s, 2H), 3.36 (s, 3H),
2.88 (s, 2H), 2.64 (s, 4H). 13C NMR (75 MHz, DMSO‐d6): δ = 165.11,
152.02, 147.22, 147.18, 145.14, 143.19, 141.04, 135.08, 133.00,
129.67, 127.96, 127.17, 126.31, 124.53, 121.32, 120.24, 114.90,
112.52, 111.63, 111.01, 101.07, 56.35, 56.17, 56.08, 56.00, 55.99,
54.58, 50.81, 48.90, 36.64, 28.77, and 17.76. ESI–MS m/z: 587.6
[M+H]+. Anal. calcd. for C32H38N6O5: C, 65.51; H, 6.53; N, 14.32;
Found: C, 65.47; H, 6.51; N, 14.28.
N‐(2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐
1H‐1,2,3‐triazol‐4‐yl)methyl)amino)phenyl)‐3,4‐dinitrobenzamide
(14)
Yield: 73.2%; brown powder; mp: 151–153℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 10.35 (s, 1H), 9.19 (s, 2H), 9.01 (s, 1H), 7.94 (s, 1H),
7.15 (d, J = 7.4 Hz, 1H), 7.11–6.99 (m, 1H), 6.85–6.51 (m, 4H), 5.88 (s,
1H), 4.51 (s, 2H), 4.37 (d, J = 4.6 Hz, 2H), 3.70 (d, J = 17.6 Hz, 6H), 3.51
(s, 2H), 2.86 (s, 2H), 2.63 (s, 4H). 13C NMR (75 MHz, DMSO‐d6):
δ = 164.45, 147.22, 147.18, 144.30, 142.68, 141.01, 138.81, 137.96,
131.00, 128.18, 127.17, 126.71, 126.60, 125.92, 123.72, 122.39,
121.44, 121.32, 115.78, 112.52, 111.01, 56.16, 56.00, 55.99, 54.58,
50.65, 48.90, 36.52, and 28.76. ESI–MS m/z: 603.5 [M+H]+. Anal.
calcd. for C29H30N8O7: C, 57.80; H, 5.02; N, 18.60; Found: C, 57.73;
H, 5.06; N, 18.54.
2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐1H‐
1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐dimethoxy‐N‐(3,4,5‐trimethoxy-
phenyl)benzamide (11)
Yield: 58.6%; brown powder; mp: 148–150℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.74 (s, 1H), 8.03 (d, J = 4.1 Hz, 2H), 7.32 (s, 1H), 7.07
(d, J = 3.9 Hz, 2H), 6.66–6.54 (m, 2H), 6.47 (s, 1H), 4.60–4.39 (m, 4H),
3.83–3.69 (m, 12H), 3.52 (s, 2H), 2.88 (s, 2H), 2.63 (s, 4H). 13C NMR
(75 MHz, DMSO‐d6): δ = 165.45, 154.18, 152.33, 147.19, 147.14,
145.23, 143.25, 141.02, 135.33, 133.24, 127.27, 126.55, 121.31,
114.81, 112.47, 111.60, 111.54, 101.28, 101.09, 60.78, 56.37, 56.34,
56.16, 56.01, 55.99, 54.59, 50.65, 48.86, 36.64, and 28.78. ESI–MS
m/z: 663.6 [M+H]+. Anal. calcd. for C34H42N6O8: C, 61.62; H, 6.39; N,
12.68; Found: C, 61.58; H, 6.35; N, 12.67.
N‐(2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐
1H‐1,2,3‐triazol‐4‐yl)methyl)amino)phenyl)‐4‐isocyanobenzamide
(
15
)
Yield: 68.4%; brown powder; mp: 155–157℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.90 (s, 1H), 8.13 (d, J = 8.0 Hz, 2H), 8.00 (d, J = 7.9 Hz,
2H), 7.92 (s, 1H), 7.14 (d, J = 7.4 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.74
(d, J = 8.2 Hz, 1H), 6.68–6.58 (m, 2H), 6.56 (s, 1H), 5.69 (s, 1H), 4.52
(d, J = 5.4 Hz, 2H), 4.34 (d, J = 5.5 Hz, 2H), 3.68 (d, J = 4.3 Hz, 6H), 3.50
(s, 2H), 2.85 (t, J = 5.4 Hz, 2H), 2.62 (s, 4H). 13C NMR (75 MHz,
DMSO‐d6): δ = 166.56, 165.47, 147.23, 147.18, 141.03, 138.01,
137.98, 128.46, 128.12, 127.25, 126.35, 126.23, 124.30, 123.73,
122.37, 121.49, 121.33, 115.79, 112.52, 111.02, 56.08, 56.07, 56.03,
54.58, 50.81, 48.90, 36.52, and 28.80. ESI–MS m/z: 538.5 [M+H]+.
Anal. calcd. for C30H31N7O3: C, 67.02; H, 5.81; N, 18.24; Found: C,
67.08; H, 5.84; N, 18.26.
N‐(4‐(tert‐Butyl)phenyl)‐2‐(((1‐(2‐(6,7‐dimethoxy‐3,4‐dihydroisoqui-
nolin‐2(1H)‐yl)ethyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)amino)‐4,5‐di-
methoxybenzamide (12
)
Yield: 71.1%; brown powder; mp: 158–159℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.53 (s, 1H), 8.23 (d, J = 5.3 Hz, 1H), 8.02 (s, 1H), 7.43
(s, 1H), 7.31–7.11 (m, 4H), 6.60 (d, J = 12.6 Hz, 2H), 6.46 (s, 1H), 4.54
(t, J = 6.0 Hz, 2H), 4.41 (d, J = 5.2 Hz, 2H), 3.78–3.67 (m, 12H), 3.53 (s,
2H), 2.88 (t, J = 6.0 Hz, 2H), 2.64 (s, 4H), 2.20 (s, 3H). 13C NMR
(75 MHz, DMSO‐d6): δ = 165.11, 152.14, 147.21, 147.17, 146.10,
145.47, 143.24, 141.02, 136.98, 127.27, 126.55, 125.55, 121.31,
120.51, 114.81, 112.47, 111.61, 111.01, 101.09, 56.35, 56.16, 56.00,
55.99, 54.59, 50.65, 48.86, 36.64, 34.55, 31.20, and 28.78. ESI–MS
2‐Chloro‐N‐(2‐(((1‐(2‐(6,7‐dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐
yl)ethyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)amino)phenyl)benzamide (16
)
Yield: 63.2%; brown powder; mp: 148–150℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.76 (s, 1H), 7.96 (d, 1H), 7.68–7.57 (m, 1H), 7.56–7.37

QIU ET AL.
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(m, 3H), 7.30 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 6.8 Hz, 1H), 6.76 (d,
J = 7.8 Hz, 1H), 6.71–6.48 (m, 3H), 4.64–4.26 (m, 4H), 3.84–3.58 (m,
6H), 3.51 (s, 2H), 2.86 (s, 2H), 2.63 (s, 4H). 13C NMR (75 MHz, DMSO‐
d6): δ = 164.88, 147.23, 147.18, 141.04, 137.98, 134.27, 132.02,
131.92, 130.01, 129.52, 127.75, 127.25, 126.49, 126.35, 123.70,
122.35, 121.49, 121.33, 115.79, 112.52, 111.02, 56.08, 56.07, 56.03,
54.58, 50.86, 48.90, 36.52, and 28.93. ESI–MS m/z: 548.1 [M+H]+.
Anal. calcd. for C29H31ClN6O3: C, 63.67; H, 5.71; N, 15.36; Found: C,
63.64; H, 5.73; N, 15.34.
calculated using GraphPad Prism 6.0 software from the dose–
response curves.
|
4.2.2
Reversing DOX resistance assay
K562 and K562/A02 cells (1 × 105) were seeded into 96‐well plates
in RPMI‐1640 and incubated for 24 hr. The target compound was
prepared as 5 mM DMSO stocks, the cells were incubated in the
presence of DOX with or without P‐gp inhibitors for 48 hr. Then,
MTT was added directly to the cells. After additional incubation for
4 hr at 37°C, the absorbance at 490 nm was read on a microplate
reader (Thermo Fisher Scientific). The IC50 values of the compounds
for cytotoxicity were calculated from the dose−response curves by
using GraphPad Prism 5.0 software (GraphPad Software, San Diego,
CA). Cell inhibition rate = 1 − (average OD of experimental group/
average OD of the control group) × 100%. Reversal fold (RF) = IC50A/
IC50B. IC50A refers to IC50 of doxorubicin towards K562/A02 cells
with no reversal agents existing. IC50B refers to IC50 of doxorubicin
towards K562/A02 cells with reversal agents existing.
N‐(2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐
1H‐1,2,3‐triazol‐4‐yl)methyl)amino)phenyl)‐2‐methylbenzamide (17)
Yield: 74.1%; brown powder; mp: 156–157℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.62 (s, 1H), 7.99 (d, J = 4.2 Hz, 1H), 7.55 (s, 1H),
7.47–7.20 (m, 4H), 7.03 (s, 1H), 6.77 (s, 1H), 6.70–6.52 (m, 3H),
4.63–4.29 (m, 4H), 3.68 (d, J = 4.0 Hz, 6H), 3.51 (s, 2H), 2.87 (d,
J = 3.8 Hz, 2H), 2.63 (s, 4H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO‐
d6): δ = 165.92, 147.23, 147.18, 141.04, 137.98, 136.56, 133.71,
129.99, 129.21, 128.35, 127.75, 127.17, 126.31, 125.89, 123.73,
122.34, 121.49, 121.33, 115.79, 112.52, 111.02, 56.08, 56.05, 56.02,
54.58, 50.81, 48.90, 36.52, 28.96, and 19.98. ESI–MS m/z: 527.6
[M+H]+. Anal. calcd. for C30H34N6O3: C, 68.42; H, 6.51; N, 15.96;
Found: C, 68.45; H, 6.53; N, 15.92.
|
4.2.3
Duration of MDR reversal effect
K562/A02 cells (1 × 105 per well) were placed into 96‐well plates in
RPMI‐1640 and incubated overnight. Then, the cells were incubated
for 24 hr with compound 11 or VRP or none at the concentration of
5 µM being washed 0 or three times with growth medium. Then the
cells were incubated for 0, 6, 12 or 24 hr before the addition of
different concentrations of DOX or the vehicle. The incubation
continued for 48 hr before MTT analysis. The IC50 values of the
compounds for cytotoxicity were calculated by GraphPad Prism 6.0,
software from dose–response curves.
N‐(2‐(((1‐(2‐(6,7‐Dimethoxy‐3,4‐dihydroisoquinolin‐2(1H)‐yl)ethyl)‐
1H‐1,2,3‐triazol‐4‐yl)methyl)amino)phenyl)‐2‐nitrobenzamide (18)
Yield: 59.8%; brown powder; mp: 148–149℃. 1H NMR (300 MHz,
DMSO‐d6): δ = 9.97 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.00–7.81 (m, 3H),
7.81–7.69 (m, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.09–6.95 (m, 1H), 6.75 (d,
J = 8.1 Hz, 1H), 6.67–6.44 (m, 3H), 4.61–4.26 (m, 4H), 3.68 (d,
J = 4.2 Hz, 6H), 3.50 (s, 2H), 2.86 (s, 2H), 2.56 (s, 4H). 13C NMR
(75 MHz, DMSO‐d6): δ = 164.68, 147.23, 147.18, 146.05, 141.03,
137.97, 131.37, 130.66, 130.59, 129.52, 127.75, 127.25, 126.35,
124.01, 123.71, 122.37, 121.44, 121.33, 115.79, 112.52, 111.02,
56.08, 56.04, 56.01, 54.58, 50.86, 48.90, 36.52, and 28.79. ESI–MS
m/z: 558.6 [M+H]+. Anal. calcd. for C29H31N7O5: C, 62.47; H, 5.60; N,
17.58; Found: C, 62.44; H, 5.64; N, 17.56.
|
4.2.4
DOX accumulation on K562/A02 cells
K562 and K562/A02 cells (1 × 105) were incubated with 20.0 μM
DOX and different concentrations of the tested compound and VRP
for 150 min at 37°C, with 0.1% DMSO as a negative control. After
incubation, the cells were washed with cold PBS and lysed with lysis
buffer (0.75 M HCl, 0.2% Triton‐X 100 in 2‐propanol). The fluores-
|
4.2
Biological evaluation
Cytotoxicity assay
cence level of DOX in the lysate was measured by using
a
|
4.2.1
fluorescence spectrophotometer (RF‐5301 PC, Shimadzu) using an
Initially, 1 × 104 K562 and K562/A02 cells were seeded into 96‐well
plates in RPMI‐1640 and incubated for 24 hr. In the assay for
cytotoxicity, a graded dose of the compounds diluted with the
medium were added into cells. In the assay for drug‐resistant
modulation, 4 µM concentrations of the target compounds were
added into cells, followed by the numerous concentrations of DOX.
The cells were incubated for another 48 hr 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 hr at 37°C, the optical density (OD)
excitation and an emission wavelength pair of 460 and 587 nm.
|
4.2.5
Rhodamine‐123 efflux assay
K562/A02 cells (1 × 105) were incubated with medium containing
5 μM Rh123 at 37°C for 90 min, washed three times with Rh123‐free
medium, and then incubated in the presence or absence of different
concentrations of compound 11, VRP, at 37°C for 5, 10, 25, 30, 60,
and 90 min, respectively. The mean fluorescence intensity (MFI) of
the retained Rh123 in per 10,000 cells was measured by flow
cytometry. Graphs were plotted of cell‐associated MFI against time.
at 490 nm was read on
a microplate reader (Thermo Fisher
Scientific). The IC50 value of the compounds for cytotoxicity was

10 of 10
QIU ET AL.
|
ACKNOWLEDGMENTS
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Foundation of Jiangsu Provincial Key Laboratory of Coastal Wetland
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