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

Article abstract of DOI:10.1016/j.bmc.2014.10.032

A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthesized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards GES-1 cells (IC₅₀ >80 μM) and K562/A02 cells (IC₅₀ >80 μM) exhibited more potency than verapamil (VRP) on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamycin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions, which may be a promising P-gp-mediated MDR modulator for further development.

Full text of DOI:10.1016/j.bmc.2014.10.032

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of novel triazole core based
P-glycoprotein-mediated multidrug resistance reversal agents
a,
Lei Jiao a, Qianqian Qiu a, Baomin Liu a,b, Tianxiao Zhao a, Wenlong Huang a, , Hai Qian
⇑
⇑
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Nan Jing Research and Development Center, CTTQ Pharmaceutical Research Institute, Building 9, NO. 699-8, Xuanwu Dadao, Xuanwu District, Nanjing, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of triazol-N-ethyl-tetrahydroisoquinoline based compounds were designed and synthe-
sized via click chemistry. Most of the synthesized compounds showed P-glycoprotein (P-gp)-mediated
multidrug resistance (MDR) reversal activities. Among them, compound 7 with little cytotoxicity towards
Received 25 September 2014
Revised 22 October 2014
Accepted 23 October 2014
Available online xxxx
GES-1 cells (IC50 >80 lM) and K562/A02 cells (IC50 >80 lM) exhibited more potency than verapamil (VRP)
on increasing anticancer drug accumulation in K562/A02 cells. Moreover, compound 7 could significantly
reverse MDR in a dose-dependent manner and also persist longer chemo-sensitizing effect than VRP with
reversibility. Further mechanism studies revealed that compound 7 in reversing MDR revealed that it
could remarkably increase the intracellular accumulation of both rhodamine-123 (Rh123) and adriamy-
cin (ADM) in K562/A02 cells as well as inhibit their efflux from the cells. These results suggested that
compound 7 showed more potency than the classical P-gp inhibitor VRP under the same conditions,
which may be a promising P-gp-mediated MDR modulator for further development.
Keywords:
Click chemistry
Multidrug resistance
P-glycoprotein
Reversal activity
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
For designing novel multidrug resistance reversal agents, an
approach we adopted was to connect chemical fragments, such
Multidrug resistance (MDR) is a serious obstruction for success-
ful tumor chemotherapy in the clinic.1 It occurs due to the over-
expression of membrane-associated transport proteins named
ATP-binding cassette (ABC) family including P-glycoprotein (P-gp,
ABCB1), the breast cancer-resistance protein (BCRP, ABCG2) and
the multidrug resistance-associated protein 1 (MRP1, ABCC1),
which has been recognized contributing to the decreased intracel-
lular concentrations of chemotherapeutic drugs.2 Among them, the
most intensive study is focused on P-gp, which has the ability to
transport a wide variety of structurally unrelated compounds out
of tumor cells resulting in MDR.3,4 Obviously, developing P-gp
inhibitors to avoid P-gp-mediated MDR should be regarded as a
potential strategy.5 During the past three decades, considerable
efforts have been made to suppress MDR and three generations
of P-gp inhibitors have been developed such as Verapamil, Valspo-
dar (PSC-833), dexverapamil and Tariquidar (XR9576).6,7 However,
all of these P-gp inhibitors suffer from toxicity or low selectivity or
drug-drug interactions. Up to now, no P-gp inhibitors have been
approved for clinical application.8 Therefore, the exploration of
potent P-gp inhibitors with high selectivity and low toxicity for
cancer treatment remains a major goal in this field.
as N-ethyl-tetrahydroisoquinoline and aromatic amides, which
are frequently appeared in reported potent p-gp inhibitors
based on a Triazole Core by click chemistry method (Fig. 1).9–11
1,2,3-Triazole ring, a hydrophobic aromatic group, associating with
biological targets through hydrogen bonding and dipole interac-
tions was introduced to the designed compounds by click
chemistry.11 Click chemistry, commonly copper-(I)-catalyzed
1,2,3-triazole formation from azides and terminal acetylenes, has
been widely applied in drug discovery.12 Based on the principles
above, we synthesized and biologically evaluated compounds
1–19 as P-gp-mediated MDR reversal agents.
2. Chemistry
The synthetic routes of target compounds 1–19 are outlined in
Scheme 1. Compound a was prepared starting from 2-nitrophenol
and 3-bromo-prop-1-yne, which refluxed in the mixture of acetone
and potassium carbonate for 4 h. Treatment of compound a with
ammonium chloride in the presence of iron powder in 80% ethanol
afforded compound b which was then reacted with freshly formed
aromatic acyl chloride and triethylamine in dry dichloromethane
to give compounds 1c–19c. Compounds d–f were synthesized
according to literature procedures with minor modification.13–15
Subsequently, compounds 1c–19c and compound f were treated
with ascorbate sodium and copper sulfate in 75% methanol stirring
⇑
Corresponding authors. Tel.: +86 25 83271302; fax: +86 25 83271480.
E-mail addresses: ydhuangwenlong@126.com (W. Huang), qianhai24@163.com
(H. Qian).
http://dx.doi.org/10.1016/j.bmc.2014.10.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. (A) Structures of the reported potent P-gp inhibitors containing chemical fragments such as N-ethyltetrahydroisoquinoline and aromatic amides. (B) Design of the
target compounds 1–19.
Scheme 1. Synthesis of the target compounds. Reagents and conditions: (i) 3-bromoprop-1-yne, K2CO3, acetone, reflux, 4 h; (ii) Fe/NH4Cl, 80% EtOH, reflux, 5 h; (iii) aromatic
acyl chlorides, TEA/DCM, rt, 24 h; (iv) NaN3, water, 80 °C, 24 h; (v) TEA/DCM, TsCl, rt, 24 h; (vi) TEA/acetonitrile, 60 °C, 24 h; (vii) 1c–19c, ascorbate sodium, CuSO4, 75%
CH3OH, 24–48 h.
at room temperature for 24–48 h to provide compounds 1–19.
Interestingly, some of the target compounds could precipitate from
the reaction solution directly and were isolated with high purity.
While others which could not precipitate directly were purified
by column chromatography. The structures of target compounds
obtained were listed in Table 1.
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L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Table 1
Structures and ADM-resistance reversal activity of the target compounds 1–19 at
M concentration in K562/A02 cellsa
Table 1 (continued)
Compounds
R0
IC50 of ADM (lM)
RF
5
l
N
O
N
N
17
5.21 0.52
10.4
O
O
N
N
N
NH
R'
18
8.89 0.55
6.1
2.9
O
Compounds
R0
IC50 of ADM (
l
M)
RF
19
18.86 1.65
N
1
2
10.81 1.45
5.0
Controlb
VRP
54.22 2.15
6.80 0.45
1
8.7
a
6.75 0.11
7.16 0.18
8.1
7.6
The IC50 value was determined after exposure to a series of ADM concentration
with different target compounds at 5 M using K562/A02 cells. Reversal fold (RF)
refers to fold-change in drug sensitivity. RF = (IC50 without modulator)/(IC50with
OCH3
l
5
lM modulator).
b
0.1% DMSO was added as solvent control for testing the P-gp modulating
3
activity.
CH3
3. Results and discussion
4
5
6
7
9.71 0.66
9.32 0.67
6.45 0.80
1.67 0.48
5.6
5.8
3.1. Biological evaluation
OCH3
N
3.1.1. Effects of the target compounds on reversing ADM
resistance in K562/A02 cells
The effects of the target compounds on reversing adriamycin
(ADM) resistance towards human erythroleukemia adriamycin-
selected K562/A02 cells (P-gp-overexpression) were preliminarily
investigated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-
H-tetrazolium bromide (MTT) method.16,17 The well-known
classical P-gp inhibitor VRP was chose as a positive control. All
8.4
O
O
compounds 1–19 and verapamil were assayed at 5 lM, a low con-
32.5
centration which could result in less than 10% cytotoxicity towards
K562/A02 cells after 48 h incubation (data not shown). As the
results summarized in Table 1, anticancer drug ADM alone
displayed little inhibitory effect on the survival of K562/A02 cells
8
9
15.63 1.58
7.25 0.31
6.25 0.83
5.21 0.29
3.5
7.5
NO2
(IC50 of 54.22 2.15 lM). However, the combination treatment of
ADM with target compounds or VRP increased the inhibitory effect
in different degrees. It revealed that most of the target compounds
exerted MDR reversal activities. Among them, compound 7 with a
H3C
significantly decreased IC50 of ADM (1.67 0.48 lM) showed the
10
11
8.7
strongest reversal activity and its reversal fold (RF) was 32.5. In
addition, some of the target compounds exhibited more active
MDR reversal activity than the positive control VRP, when
co-administered with ADM at the same condition.
H3CO
10.4
Cl
H3CO
3.1.2. Cytotoxicity assay
12
13
14
3.23 0.45
14.43 0.03
13.85 0.31
16.8
3.6
H3CO
Based on the results above, the most potent compound 7 was
selected for further study. The intrinsic cytotoxicity of the selected
compound 7 against K562/A02 and normal human gastric epithe-
lial cell strain-1 (GES-1) was examined by MTT assay. As shown
in Figure 2, compound 7 exhibited little intrinsic cytotoxicity
OCH3
NC
H3CO
against either GES-1 (IC50 >80
>80 M). Particularly, concentrations of compound 7 ranging from
1.25 M to 80 M, showed more inhibitory effects on MDR K562/
lM) or K562/A02 cells (IC50
l
3.9
l
l
OCH3
A02 cells than on normal GES-1 cells. It suggests that compound
7 may be more secure to normal cells comparing to tumor cells.
Thus, compound 7, which has little cytotoxicity to normal cells
and P-gp overexpressing cell line, is a suitable candidate for the
development of MDR reversal agents.
15
16
11.00 1.06
15.07 0.20
4.9
3.6
Cl
3.1.3. Chemo-sensitizing effect of target compounds
To further investigate the reversal potency and dose–response
effects, we have determined the reversal activity of compound 7
NO2
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L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 3
Duration of MDR reversal in K562/A02 cells after incubation and washout of
verapamil or compound 7
Treatment
schedule
IC50/ADM (
VRP (5 M)
l
M) (RF)a
Control
l
Compound
7 (5 M)
l
No wash
80.8 4.25 (1)
9.60 2.23 (8.4)
40.80 3.71 (1.9)
>80
2.16 0.16 (37.4)
10.97 1.53 (7.7)
35.61 2.30 (2.4)
54.53 1.30 (1.5)
Wash, 0 h
Wash, 6 h
Wash, 12 h
ndb
nd
nd
>80
a
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.
b
nd: not determined.
Figure 2. Effect of Compound 7 on the growth of K562/A02 cells and GES-1 cells.
The cells were treated with various concentrations (0, 1.25, 2.5, 5, 10, 20, 40, 60,
80 lM) of compound 7 for 48 h. Viable cells were evaluated by MTT assay. Data are
expressed as mean SD of three independent experiments.
Table 2
Sensitization of compound 7 on reversing MDR towards K562/A02 cells at different
concentrationsa
Compounds
None
IC50 of ADM (
lM)
RF
1
54.93 2.37
15.92 1.17
2.97 0.16
3.14 0.30
5.20 0.35
6.39 0.41
37.03 3.4
43.8 0.16
32.94 1.04
VRP, 2.5
Compound 7, 2.5
Compound 7, 1.25
Compound 7, 0.625
lM
3.4
l
l
M
M
l
18.5
17.4
10.5
8.6
1.5
1.3
M
Compound 7, 0.31
Compound 7, 0.156
Compound 7, 0.078
lM
l
l
M
M
M
Compound 7, 0.04
l
1.6
a
Reversal fold (RF), RF = (IC50 without modulator)/(IC50 with modulator). Each
experiment was carried out two to three times, and the values were presented as
the mean standard error of mean.
Figure 3. The effects of the target compounds on the intracellular accumulation of
Rh123 (5
lM) in K562 and K562/A02 cells. Cells were pretreated with 0.5, 2.5, 5
lM
of compound 7 or VRP for 60 min and then exposed to 5
l
M of Rh123 for another
90 min. After treatment, the cells were washed twice with PBS and resuspended in
medium. Rh123-associated mean fluorescence intensity was evaluated by flow
cytometry. The Rh123 accumulation fold change values were identified by dividing
the fluorescence intensity obtained from each measurement by that of control cells
(0.1% DMSO) in each cell group, respectively. Data represent means SD of three
independent experiments. ⁄P <0.05, ⁄⁄P <0.01 versus untreated K562/A02 cells.
at various other concentrations (2.5
lM, 1.25 lM, 0.625 lM,
0.31 M, 0.156 M, 0.078 M, 0.04 M) towards K562/A02 cells
l
l
l
l
(P-gp-overexpression) by MTT assay, selecting VRP as positive con-
trols.18 As demonstrated in Table 2, VRP showed slight modulating
activity at 2.5
dependent activity and still exhibited potent MDR reversal activity
(RF = 8.6) when the concentration decreased to 0.31 mol/L. Addi-
lM, however, compound 7 showed apparent dose
towards K562/A02 cells which pretreated with VRP and compound
7, were 9.60 lM, 2.16 lM respectively (no wash group). The MDR-
l
tionally, the EC50 value of compound 7 was 147.7 5.6 nM, which
was calculated by GraphPad Prism 5.0 software from the dose–
response curves. The results suggested that compound 7 had the
significantly potential to enhance the sensitivity of P-gp-over-
expressing cells to anticancer drug substrates in a dose-dependent
manner.
reversing effects of VRP (RF = 1.9) and compound 7 (RF = 7.7)
decreased immediately after their removal from the medium.
However, the reversal effect of VRP was disappeared after its
removal from the medium for 6 h. In contrast, compound 7 showed
reversal activity even after its removal from the medium for 12 h
and the IC50 of ADM was 54.53 lM (RF = 1.5). These data indicated
that compound 7 displayed potent MDR-reversing effect and
persisted for longer time compared with the positive control
VRP. The data also indicated that the chemo-sensitizing effect of
compound 7 towards K562/A02 cells was reversible.
3.1.4. Duration of chemo-sensitizing effect of compound 7
toward ADM in K562/A02 cells
It is known that a relatively long duration of action with revers-
ibility for a P-gp inhibitor is likely to be required for safe and
effective therapy of P-gp-mediated MDR cancers.19 Therefore, the
duration of MDR reversal effect of compound 7 was evaluated
according to the procedure previously described with minor mod-
3.1.5. Effect of the target compounds on Rh123 accumulation
Furthermore, in order to investigate the mechanism of com-
pound 7 in modulating the anticancer drug substrates accumula-
tion level inside K562/A02 cells, the intracellular accumulation of
rhodamine-123 (Rh123), a fluorescence substrate of P-gp, was
assessed by monitoring its fluorescence intensity through flow
cytometry assay. It is known that P-gp transporter, which overex-
presses on the adriamycin-selected K562/A02 cells, can rapidly
increase Rh123 efflux from cells and result in a decrease of
Rh123 in the intracellular accumulation. As shown in Figure 3,
ification.19 Briefly, K562/A02 cells were incubated with 5
lM of
compound 7, VRP or vehicle for 24 h, then washed with growth
medium. Various concentrations of ADM was then added to the
culture at different time points (0, 6 or 12 h) after the removal of
the compounds, and incubated for additional 48 h. The duration
of the MDR-reversal activity of compound 7 or VRP was examined
by MTT assay. As demonstrated in Table 3, the IC50s of ADM
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L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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the classical P-gp inhibitor VRP was employed as a positive control.
Once treated with compound 7 (5 M), the P-gp-overexpressing
K562/A02 cells exhibited significantly increased accumulation of
Rh123. In comparison, both VRP and compound 7 showed no influ-
ence on the Rh123 accumulation fold change of ADM-sensitive
K562 cells. When the K562/A02 cells were treated with compound
7 at various concentrations (0.5 lM, 2.5 lM, 5 lM), the retained
amount of Rh123 was significantly increased in a dose-dependent
manner (1.55, 2.40, 4.04). Moreover, the Rh123 accumulation
after 90-min incubation. On the contrary, there was little
fluorescence of ADM being observed in P-gp overexpression
K562/A02 cells (Fig. 4 Control). It occured due to the overexpres-
sion P-gp of K562/A02 cells could efficiently pump ADM out of
the cells after a 90-min incubation. When K562/A02 cells were
treated with compound 7 and VRP, the accumulation of ADM
was significantly increased in a dose-dependent manner and the
fluorescence intensity of K562/A02 cells treated with compound
7 was higher than that of VRP at the same concentrations.
l
fold change of compound 7 (5 lM) was 4.04, which was 2.4
times greater than that of VRP to K562/A02 cells at the same
concentration. The results indicated that compound 7 exhibited
dose-dependent effects on increasing accumulation of Rh123
towards K562/A02 cells. Additionally, compound 7 which was
more potent than VRP in Rh123 accumulation might effectively
block the drug efflux function of P-gp.
3.1.7. Inhibitory effect of compound 7 on P-gp efflux function
In order to further verify the above presumption, the inhibitory
effect of compound 7 on the efflux function of P-gp was assayed by
detecting the retained intracellular Rh123 according to the method
described in literatures with minor modification.20,21 As demon-
strated in Figure 5, the sensitive K562 cells retained most of the
fluorescence Rh123, while the K562/A02 cells (P-gp overexpres-
sion) showed comparatively low intracellular level of Rh123. How-
ever, when treated with compound 7 or VRP, the Rh123 level in
K562/A02 cells was obviously increased in a dose-dependent man-
ner. Particularly, compound 7 was more potent than VRP at the
same doses. Such as, the fluorescence intensity of Rh123 in
3.1.6. Effect of the target compound 7 on ADM accumulation
To validate our assumption, we evaluated the ADM accumula-
tion, another fluorescence substrate of P-gp, towards K562/A02
cells by using fluorescence microscope. As shown in Figure 4, the
sensitive K562 cells retained most of the red fluorescence ADM
Figure 4. The effect of the target compounds on the intracellular accumulation of ADM (20
lM). K562 cells (A1) or K562/A02 cells (A2, B–C) were incubated without (control:
A) or with compound 7 (B1: 0.5 lM, B2: 5 lM) or VRP (C1: 0.5 lM, C2: 5 lM) for 60 min, then treated with 20 lM ADM at 37 °C for another 90 min. After treatment, the cells
were washed twice with PBS and centrifuged. The cells were then observed and photographed under a fluorescence microscope. VRP was used as a positive control and 0.1%
DMSO was used as vehicle control.
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L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
based on the click chemistry and evaluated in vitro as P-gp-medi-
ated MDR reversal agents. Among them, compound 7 with low
cytotoxicity could significantly reverse MDR in a dose-dependent
manner and persist longer chemo-sensitizing effect than VRP with
reversibility. Additionally, compound 7 could remarkably increase
the intracellular accumulation of both Rh123 and ADM in K562/
A02 cells as well as inhibit their efflux from the cells. These results
suggest that compound 7 could effectively block the drug efflux
function of P-gp and lead to increased drug accumulation in MDR
cells. Therefore, compound 7 could be served as a promising candi-
date for the development of P-gp-mediated MDR-Reversal Agents.
5. Experimental section
5.1. General chemistry
All reagents were reagent grade and all solvents were dried by
standards methods before using. Column chromatography was car-
ried 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 compounds were analyzed
by 1H NMR, 13C NMR (Bruker ACF-300Q, 300 MHz), MS (1100 LC/
MSD spectrometer; Hewlett–Packard) and elemental analyses
(CHN-O-Rapid instrument); 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.9,10 The starting compound
2-nitrophenol was commercially available.
Figure 5. Inhibitory effect of compound 7 on the efflux of Rh123. K562 or K562/A02
cells were incubated with 5
three times. Then the cells were incubated with or without various concentrations
of compound 7 or VRP (0.5, 2.5, 5 M) for another 90 min. Afterwards the cells were
lM Rh123 for 60 min before washing with ice PBS for
l
washed twice in ice-cold PBS. The mean fluorescence intensity of retained
intracellular Rh123 was estimated by flow cytometry. Data were expressed as
means SD of two independent experiments. ⁄P <0.05, ⁄⁄P <0.01 versus untreated
K562/A02 cells.
K562/A02 cells treated with compound 7 was 42.5, which was sig-
nificantly higher than that of VRP with 29.2 at the same dose of
5 lM. These results suggest that compound 7 could effectively
block the drug efflux function of P-gp. What is more, the potency
of compound 7 is much higher than the classical P-gp inhibitor
VRP under the same conditions.
5.2. Synthesis of 1-Nitro-2-(prop-2-yn-1-yloxy) benzene (a)
The results above verified our assumption that compound 7 was
an effective P-gp inhibitor which had the potential to enhance the
sensitivity of P-gp overexpressing drug-selected cell lines to
anticancer drug substrates by effectively blocking the drug
efflux function of P-gp and showed more potency than the classical
P-gp inhibitor VRP under the same conditions.
2-Nitrophenol (11.2 g, 80 mmol) and 3-bromo-prop-1-yne
(6.4 ml, 80 mmol) was dissolved in acetone (120 mL) and added
potassium carbonate (22.0 g, 160 mmol), then the mixture was
heated to reflux for 4 h. Then the reaction mixture was cooled to
room temperature followed by filtration and evaporated in vacuo
to get compound a (14.1 g). Yield: 99.3%. Yellow powder, mp:
66–68 °C.
3.2. Structure–activity relationships
5.3. Synthesis of 2-(Prop-2-yn-1-yloxy) aniline (b)
The analysis of structure–activity relationships (SARs) according
to the results in Table 1 indicates that different substituents in R0 of
compounds 1–19 could affect MDR reversal activities towards
K562/A02 cells: (a) Electron-donating groups in R0 might be benefi-
cial for the MDR reversal activity. For example, compounds 2, 6, 7,
10 and compound 12 which possessed electron-donating group like
–OCH3 and –C(CH3)3 showed higher MDR reversal activity than oth-
ers. (b) The substitution position of electron-donating group in R0
has an influence on the reverse activity. Such as compounds 6, 7,
10 and compound 12, which were substituted in para position,
was more potent than in other positions. (c) The size of substituent
in R0 is likely to affect MDR reversal activity. For example, compound
12 (RF = 16.8) containing 3,4,5-trimethoxy in R0 was more potent
than compound 10 which was substituted by 4-methoxy. When
3,4,5-trimethoxy was replaced by 4-tertiary butyl, a bigger size sub-
stituent, the corresponding compound 7 showed the strongest
activity (RF = 32.5). It suggested that the introduction of 4-tertiary
butyl to the molecule of compound 7 could dramatically strengthen
its binding affinity to P-gp, leading to more ADM accumulation in
K562/A02 cells.
The mixture of compound a (14.0 g, 80 mmol), ammonium
chloride (21.4 g, 400 mmol) and iron powder (13.4 g, 240 mmol)
in 80% ethanol (200 mL) was heated to reflux for 5 h. After cooling
to room temperature, the mixture was adjusted with sodium
carbonate to PH = 7–8, filtered with Celite, and evaporated in vacuo
to afford a brown residue. Dichloromethane (50 mL) was added
into the residue and the resulted solution was washed by saturated
aqueous Na2CO3 (40 mL) and brine (40 mL) in sequence. The
organic layer was dried by anhydrous Na2SO4. The solvent was
evaporated to give corresponding compound b as brown oil
(9.5 g). Yield: 82.3%.
5.4. General procedure for the preparation of 1c–19c
The freshly formed aromatic acyl chloride (5 mmol) dissolved in
dry dichloromethane (25 ml) was added dropwise to a solution of
compound b (5 mmol) and triethylamine (6 mmol) dissolved in
dichloromethane (25 mL), keeping the temperature at 0 °C for
2 h, and then, the mixture was stirred at room temperature for
24 h. The reaction solution was washed by 1 N hydrochloric acid
(3 ꢀ 30 mL), saturated aqueous Na2CO3 (3 ꢀ 30 mL), and brine
(2 ꢀ 30 mL) in sequence. The organic layer was dried by anhydrous
Na2SO4, then, filtered and evaporated to afford the desired product
compound 1c–19c.
4. Conclusions
In summary, nineteen compounds containing triazol-N-ethyl-
tetrahydroisoquinoline and aromatic amides were synthesized
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5.5. Synthesis of 2-azidoethanol (d)
164.8; ESI-MS m/z: 514.4 [M+H]+, 536.3 [M+Na]+; Anal. Calcd for
C29H31N5O4: C, 67.82; H, 6.08; N, 13.64. Found: C, 67.69; H, 6.04;
N, 13.70.
To a 100 mL round bottom flask was added 2-bromoethanol
(3.4 g, 27 mmol) and sodium azide (3.1 g, 47.7 mmol) in water
(40 mL). The mixture was stirred at 80 °C for 24 h, and then, cooled
to room temperature. The solution was extracted with ethyl
acetate (4 ꢀ 30 mL) and the organic layer was dried with sodium
sulfate overnight, then, filtered. After the removal of the solvent
under vacuum, compound d was obtained as a crude pale yellow
liquid (2.74 g), which was used in the next step directly.
5.8.2. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-2-
methoxybenzamide (2)
Yield 31.5%; white powder; mp: 180–182 °C; 1H NMR
(300 MHz, CDCl3): d = 10.51 (s, 1H, CONH), 8.64 (dd, J = 6.8 Hz,
4.0 Hz, 1H, ArH), 8.25 (dd, J = 7.8 Hz, 1.7 Hz, 1H, ArH), 7.85 (s, 1H,
NCH@C), 7.42 (ddd, J = 7.6 Hz, 1.6 Hz, 1.6 Hz, 1H, ArH), 7.05 (m,
4H, ArH), 6.80 (d, J = 8.2 Hz, 1H, ArH), 6.56 (s, 1H, ArH), 6.45 (s,
1H, ArH), 5.30 (s, 2H, OCH2), 4.67 (s, 2H, CHNCH2), 3.83 (s, 3H, –
OCH3), 3.80 (s, 3H, –OCH3), 3.66 (m, 5H, ArCH2N, –OCH3), 3.12
(m, 2H, NCH2), 2.80 (m, 4H, 2 ꢀ CH2); 13C NMR (DMSO, 75 MHz,
d ppm): 28.0, 46.9, 49.9, 55.1, 55.5, 56.5, 61.6, 109.9, 111.7,
112.1, 119.3, 120.9, 121.0, 123.5, 125.6, 125.8, 126.3, 127.9,
131.3, 133.4, 141.7, 146.9, 147.0, 147.3, 157.0, 162.0; ESI-MS m/z:
544.3 [M+H]+; Anal. Calcd for C30H33N5O5: C, 66.28; H, 6.12; N,
12.88. Found: C, 66.33; H, 6.15; N, 12.70.
5.6. Synthesis of 2-azidoethyl 4-methylbenzenesulfonate (e)
To the solution of 2-azidoethanol (2.74 g, 24 mmol) and
triethylamine (8.4 mL, 60 mmol) in dry dichloromethane (20 mL),
4-toluenesulfonylchloride (4.0 g, 21 mmol) in dry dichloromethane
(25 mL) was added drop-wise under constant stirring at 0 °C and
then the mixture was stirred at room temperature for 24 h. The
reaction mixture was washed with 1 N HCl (3 ꢀ 40 mL) and 1 N
NaHCO3 (3 ꢀ 40 mL) and brine (3 ꢀ 40 mL). The organic layer
was dried over anhydrous sodium sulfate, filtered and evaporated
under reduced pressure. The crude product was purified by silica
gel column chromatography using a mixture of ethyl acetate/pet
ether (1:15, v/v) as eluent to afford the compound e (3.1 g). As col-
orless liquid, yield: 56.3%.
5.8.3. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-
methylbenzamide (3)
Yield 19.2%; pale yellow powder; mp: 106–108 °C; 1H NMR
(300 MHz, CDCl3): d = 8.61 (s, 1H, CONH), 8.47 (dd, J = 5.0 Hz,
4.5 Hz, 1H, ArH), 7.84 (s, 1H, NCH@C), 7.72 (s, 1H, ArH), 7.65 (d,
J = 6.1 Hz, 1H, ArH), 7.35 (m, 2H, ArH), 7.06 (m, 3H, ArH), 6.56 (s,
1H, ArH), 6.45 (s, 1H, ArH), 5.30 (s, 2H, OCH2), 4.70 (s, 2H, CHNCH2),
3.86 (s, 3H, –OCH3), 3.83 (s, 3H, –OCH3), 3.66 (s, 2H, ArCH2N), 3.15
(s, 2H, NCH2), 2.81 (s, 4H, 2 ꢀ CH2), 2.42 (s, 3H, –CH3); 13C NMR
(DMSO, 75 MHz, d ppm): 20.9, 28.0, 47.1, 50.0, 54.8, 55.4, 56.6,
62.8, 109.9, 111.8, 114.0, 121.1, 123.4, 124.4, 124.5, 125.2, 125.7,
126.2, 127.8, 127.8, 128.4, 132.2, 134.5, 137.9, 142.6, 146.9,
147.2, 149.8, 164.8; ESI-MS m/z: 528.3 [M+H]+, 550.6 [M+Na]+;
Anal. Calcd for C30H33N5O4: C, 68.29; H, 6.30; N, 13.27. Found: C,
68.18; H, 6.27; N, 13.31.
5.7. Synthesis of 2-(2-azidoethyl)-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline (f)
To a solution of 2-azidoethyl-4-methylbenzenesulfonate (3.1 g,
13 mmol) in dry acetonitrile (50 mL) and triethylamine (2.8 mL,
21 mmol), 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochlo-
ride (2.5 g, 11 mmol) was added. The mixture was refluxed for
24 h, evaporated in vacuo, and chromatographed on a silica gel col-
umn (EtOAc/PE 3/2 to EtOAc/MeOH 16/1) to give compound f
(2.0 g).As pale yellow solid, yield:70%.
5.8. General procedure for the preparation of compounds 1–19
5.8.4. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-3-
methoxybenzamide (4)
To a 100 mL round bottom flask was added 1c–19c (1 mmol)
and f (1 mmol) in 75% methanol (30 mL), ascorbate sodium
(23 mg) and CuSO4 (7.5 mg) were added, respectively. The reaction
solution was stirred at room temperature for 24–48 h. Some of the
reaction solutions precipitated white solid, the others none. The
solution with precipitate appeared filtered directly and the solu-
tion without precipitate appeared evaporated in vacuo, extracted
with dichloromethane. Then, the organic layer was dried by anhy-
drous Na2SO4. After filtration, the solvent was evaporated and the
crude product was purified by silica gel column chromatography
using a mixture of ethyl acetate/ Methanol (15:1, v/v) as eluent
to give the desire white solid product compounds 1–19.
Yield 18.5%; white powder; mp: 115–118 °C; 1H NMR (300 MHz,
CDCl3): d = 8.62 (s, 1H, CONH), 8.47 (dd, J = 5.1 Hz, 4.3 Hz, 1H, ArH),
7.78 (s, 1H, NCH@C), 7.38 (m, 3H, ArH), 7.06 (m, 4H, ArH), 6.56 (s,
1H, ArH), 6.46 (s, 1H, ArH), 5.30 (s, 2H, OCH2), 4.58 (t, J = 4.2 Hz,
2H, CHNCH2), 3.86 (s, 3H, –OCH3), 3.84 (s, 3H, –OCH3), 3.81 (s, 3H,
–OCH3), 3.64 (s, 2H, ArCH2N), 3.01 (s, 2H, NCH2), 2.74 (s, 4H,
2ꢀCH2); 13C NMR (DMSO, 75 MHz, d ppm): 27.9, 47.1, 50.0, 54.8,
55.3, 55.4, 56.6, 62.6, 109.9, 111.8, 112.4, 113.8, 117.5, 119.4,
121.0, 123.9, 124.5, 125.5, 125.7, 126.2, 127.5, 129.7, 135.9, 142.6,
146.9, 147.2, 150.0, 159.3, 164.5; ESI-MS m/z: 544.4 [M+H]+, 566.1
[M+Na]+; Anal. Calcd for C30H33N5O5: C, 66.28; H, 6.12; N, 12.88.
Found: C, 66.25; H, 6.14; N, 12.81.
5.8.1. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)benzamide (1)
5.8.5. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-4-
(dimethylamino)benzamide (5)
Yield 58.8%; white powder; mp: 114–116 °C; 1H NMR
(300 MHz, DMSO-d6): d = 9.40 (s, 1H, CONH), 8.18 (s, 1H, NCH@C),
7.92 (d, J = 7.1 Hz, 2H, ArH), 7.82 (d, J = 7.4 Hz, 1H, ArH), 7.53 (m,
3H, ArH), 7.29 (d, J = 7.8 Hz, 1H, ArH), 7.14 (dd, J = 7.4 Hz, 7.4 Hz,
1H, ArH), 6.99 (dd, J = 7.4 Hz, 7.4 Hz, 1H, ArH), 6.61 (s, 1H, ArH),
6.56 (s, 1H, ArH), 5.24 (s, 2H, OCH2), 4.55 (t, J = 6.0 Hz, 2H,
CHNCH2), 3.68 (s, 3H, –OCH3), 3.67 (s, 3H, –OCH3), 3.50 (s, 2H,
ArCH2N), 2.85 (t, J = 6.1 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2). 13C
NMR (DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6,
62.6, 109.8, 111.8, 113.9, 121.0, 123.8, 124.5, 125.4, 125.7, 126.2,
127.3, 127.6, 128.5, 131.6, 134.5, 142.6, 146.9, 147.1, 150.0,
Yield 71.9%; white powder; mp: 143–144 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.20 (s, 1H, CONH), 8.18 (s, 1H, NCH@C), 7.89 (d,
J = 7.0 Hz, 1H, ArH), 7.28 (dd, J = 7.9 Hz, 7.9 Hz, 2H, ArH), 7.13 (m,
3H, ArH), 7.00(dd, J = 7.5 Hz, 7.5 Hz, 1H, ArH), 6.90 (d, J = 7.4 Hz,
1H, ArH), 6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.23 (s, 2H, OCH2),
4.55 (t, J = 5.8 Hz, 2H, CHNCH2), 3.68 (s, 3H, –OCH3), 3.67(s, 3H,
–OCH3), 3.50 (s, 2H, ArCH2N), 2.92(s, 6H, 2ꢀCH3), 2.85 (t,
J = 5.9 Hz, 2H, NCH2), 2.61 (s, 4H, 2ꢀCH2); 13C NMR (DMSO,
75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.5, 109.9,
Please cite this article in press as: Jiao, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.032

8
L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
110.6, 111.8, 113.6, 114.6, 115.3, 121.0, 123.0, 124.6, 125.0, 125.7,
126.2, 127.8, 129.1, 135.2, 142.5, 146.9, 147.2, 149.4, 150.3, 165.3;
ESI-MS m/z: 557.4 [M+H]+, 579.2 [M+Na]+; Anal. Calcd For
NCH@C), 7.83 (d, J = 8.0 Hz, 3H, ArH), 7.30 (dd, J = 8.0 Hz, 8.0 Hz,
3H, ArH), 7.13 (dd, J = 6.9 Hz, 6.9 Hz, 1H, ArH), 6.98 (dd,
J = 7.4 Hz, 7.4 Hz, 1H, ArH), 6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH),
5.24 (s, 2H, OCH2), 4.55 (t, J = 5.9 Hz, 2H, CHNCH2), 3.68 (s, 3H, –
OCH3), 3.67 (s, 3H, –OCH3), 3.50 (s, 2H, ArCH2N), 2.85 (t,
J = 5.9 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2), 2.37 (s, 3H, –CH3); 13C
NMR (DMSO, 75 MHz, d ppm): 20.9, 28.0, 47.1, 50.0, 54.8, 55.4,
56.6, 62.7, 109.8, 111.7, 113.9, 121.0, 123.5, 124.5, 125.2, 125.7,
126.2, 127.3, 127.8, 129.0, 131.7, 141.6, 142.6, 146.9, 147.2,
149.8, 164.6; ESI-MS m/z: 528.4 [M+H]+, 550.3 [M+Na]+; Anal.
Calcd for C30H33N5O4: C, 68.29; H, 6.30; N, 13.27. Found: C,
68.33; H, 6.28; N, 13.25.
C31H36N6O4: C, 66.89; H, 6.52; N, 15.10. Found: C, 66.79; H, 6.55;
N, 15.15.
5.8.6. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-3,4-
dimethoxybenzamide (6)
Yield 34.9%; white powder; mp: 146–148 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.26 (s, 1H, CONH), 8.16 (s, 1H, NCH@C), 7.81 (d,
J = 7.2 Hz, 1H, ArH), 7.54 (dd, J = 8.3 Hz, 7.9 Hz, 1H, ArH), 7.48 (s,
1H, ArH), 7.28 (d, J = 7.8 Hz, 1H, ArH), 7.13 (dd, J = 6.7 Hz, 6.7 Hz,
1H, ArH), 7.05 (d, J = 8.4 Hz, 1H, ArH), 6.98 (dd, J = 7.3 Hz, 7.3 Hz,
1H, ArH), 6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.23 (s, 2H, OCH2),
4.54 (t, J = 5.9 Hz, 2H, CHNCH2), 3.82 (s, 3H, –OCH3), 3.81 (s, 3H,
–OCH3), 3.68 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3),3.49 (s, 2H, ArCH2-
N), 2.85 (t, J = 5.8 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2); 13C NMR
(DMSO, 75 MHz, d ppm): 27.9, 47.1, 50.0, 54.8, 55.4, 55.5, 55.6,
56.6, 62.6, 109.8, 110.5, 110.5, 111.0, 111.7, 113.9, 120.6, 121.1,
123.6, 124.5, 125.1, 125.7, 126.2, 126.7, 127.9, 142.6, 146.9,
147.1, 148.4, 149.8, 151.6, 164.2; ESI-MS m/z: 574.4 [M+H]+,
596.6 [M+Na]+; Anal. Calcd for C31H35N5O6: C, 64.91; H, 6.15; N,
12.21. Found: C, 64.80; H, 6.17; N, 12.25.
5.8.10. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-4-
methoxybenzamide (10)
Yield 74.1%; white powder; mp: 166–168 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.24 (s, 1H, CONH), 8.17 (s, 1H, NCH@C), 7.92 (d,
J = 8.7 Hz, 2H, ArH), 7.83 (d, J = 7.1 Hz, 1H, ArH), 7.28 (d,
J = 8.0 Hz, 1H, ArH), 7.12 (dd, J = 7.1 Hz, 7.1 Hz, 1H, ArH), 7.00 (m,
3H, ArH), 6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.25 (s, 2H, OCH2),
4.55 (t, J = 5.8 Hz, 2H, CHNCH2), 3.83 (s, 3H, –OCH3), 3.68 (s, 6H,
2 ꢀ –OCH3), 3.50 (s, 2H, ArCH2N), 2.85 (s, 2H, NCH2), 2.61 (s, 4H,
2 ꢀ CH2); 13C NMR (DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0,
54.8, 55.3, 55.4, 56.6, 62.7, 109.8, 111.7, 113.7, 113.8, 121.0,
123.6, 124.5, 125.1, 125.7, 126.2, 126.6, 127.9, 129.2, 146.9,
147.1, 149.8, 161.9, 164.2; ESI-MS m/z: 544.3 [M+H]+, 566.2
[M+Na]+; Anal. Calcd For C30H33N5O5: C, 66.28; H, 6.12; N, 12.88.
Found: C, 66.30; H, 6.11; N, 12.86.
5.8.7. 4-(tert-Butyl)-N-(2-((1-(2-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2, 3-triazol-4-
yl)methoxy)phenyl)benzamide (7)
Yield 52.7%; white powder; mp: 126–128 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.30 (s, 1H, CONH), 8.18 (s, 1H, NCH@C), 7.86 (d,
J = 8.1 Hz, 3H, ArH), 7.52 (d, J = 8.2 Hz, 2H, ArH), 7.28 (d,
J = 8.0 Hz, 1H, ArH), 7.13 (dd, J = 7.0 Hz, 7.0 Hz, 1H, ArH), 6.98
(dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH), 6.61 (s, 1H, ArH), 6.54 (s, 1H,
ArH), 5.24 (s, 2H, OCH2), 4.55 (t, J = 5.3 Hz, 2H, CHNCH2), 3.68 (s,
3H, –OCH3), 3.66 (s, 3H, –OCH3), 3.49 (s, 2H, ArCH2N), 2.85 (t,
J = 5.7 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2), 1.31 (s, 9H, 3ꢀCH3);
13C NMR (DMSO, 75 MHz, d ppm): 28.0, 30.9, 34.6, 47.1, 50.0,
54.8, 55.4, 56.6, 62.6, 109.8, 111.7, 113.8, 121.0, 123.4, 124.5,
125.2, 125.3, 125.7, 126.1, 127.2, 127.7, 131.7, 142.6, 146.9,
147.1, 147.1, 149.7, 154.5, 164.6; ESI-MS m/z: 570.2 [M+H]+,
592.5 [M+Na]+; Anal. Calcd for C33H39N5O4: C, 69.57; H, 6.90; N,
12.29. Found: C, 69.62; H, 6.88; N, 12.33.
5.8.11. 4-Chloro-N-(2-((1-(2-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)benzamide (11)
Yield 40.0%; yellow powder; mp: 150–153 °C; 1H
NMR(300 MHz, DMSO-d6): d = 9.54 (s, 1H, CONH), 8.16 (s, 1H,
NCH@C), 7.95 (d, J = 8.4 Hz, 2H, ArH), 7.74 (d, J = 7.5 Hz, 1H, ArH),
7.58 (d, J = 8.4 Hz, 2H, ArH), 7.28 (d, J = 8.1 Hz, 1H, ArH), 7.16 (dd,
J = 7.1 Hz, 7.1 Hz, 1H, ArH), 7.00 (dd, J = 7.5 Hz, 7.5 Hz, 1H, ArH),
6.61 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.23 (s, 2H, OCH2), 4.55 (t,
J = 5.7 Hz, 2H, CHNCH2), 3.68 (s, 6H, 2 ꢀ –OCH3), 3.50 (s, 2H, ArCH2-
N), 2.85 (t, J = 5.9 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2); 13C NMR
(DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.7,
109.9, 111.8, 114.0, 121.0, 124.5, 125.7, 126.2, 127.4, 128.5,
129.4, 133.3, 136.4, 142.7, 146.9, 147.2, 150.4, 164.0; ESI-MS m/z:
548.3 [M+H]+, 570.2 [M+Na]+; Anal. Calcd for C29H30ClN5O4: C,
63.56; H, 5.52; Cl, 6.47; N, 12.78. Found: C, 63.49; H, 5.53; Cl,
6.49; N, 12.77.
5.8.8. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-2-
nitrobenzamide (8)
Yield 71.7%; yellow powder; mp: 149–152 °C; 1H
NMR(300 MHz, DMSO-d6): d = 9.91 (s, 1H, CONH), 8.15 (s, 1H,
NCH@C), 8.12 (d, J = 8.0 Hz, 1H, ArH), 7.75 (m, 4H, ArH), 7.25 (d,
J = 8.1 Hz, 1H, ArH), 7.13 (dd, J = 7.2 Hz, 7.2 Hz, 1H, ArH), 6.98
(dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH), 6.62 (s, 1H, ArH), 6.56 (s, 1H,
ArH), 5.21 (s, 2H, OCH2), 4.55 (t, J = 5.7 Hz, 2H, CHNCH2), 3.68 (s,
3H, –OCH3), 3.67 (s, 3H, –OCH3), 3.51 (s, 2H, ArCH2N), 2.87 (t,
J = 5.8 Hz, 2H, NCH2), 2.63 (s, 4H, 2 ꢀ CH2); 13C NMR (DMSO,
75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.3, 109.9,
111.8, 113.6, 120.8, 123.9, 124.1, 124.7, 125.6, 125.7, 126.2,
127.1, 129.2, 130.7, 132.8, 133.9, 142.5, 146.5, 146.9, 147.2,
149.8, 164.4; ESI-MS m/z: 559.3 [M+H]+, 581.1 [M+Na]+; Anal.
Calcd for C29H30N6O6: C, 62.36; H, 5.41; N, 15.05. Found: C,
62.42; H, 5.44; N, 15.01.
5.8.12. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-3,4,5-
trimethoxybenzamide (12)
Yield 66.3%; white powder; mp: 124–126 °C; 1H NMR
(300 MHz, DMSO-d6): d = 9.36 (s, 1H, CONH), 8.16 (s, 1H, NCH@C),
7.78 (d, J = 7.4 Hz, 1H, ArH), 7.30 (d, J = 8.0 Hz, 1H, ArH), 7.23 (s, 2H,
ArH), 7.16 (dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH), 7.00 (dd, J = 7.7 Hz,
7.7 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 6.55 (s, 1H, ArH), 5.22 (s, 2H,
OCH2), 4.54 (t, J = 5.7 Hz, 2H, CHNCH2), 3.83 (s, 6H, 2 ꢀ –OCH3),
3.68 (m, 9H, 3 ꢀ –OCH3), 3.49 (s, 2H, ArCH2N), 2.85 (t, J = 5.6 Hz,
2H, NCH2), 2.61 (s, 4H, 2ꢀCH2); 13C NMR (DMSO, 75 MHz, d
ppm): 27.9, 47.1, 50.0, 54.8, 55.4, 55.9, 56.6, 60.1, 62.6, 104.8,
109.8, 111.7, 113.8, 121.1, 123.9, 124.6, 125.4, 125.7, 126.2,
127.6, 129.7, 129.7, 142.5, 146.9, 147.2, 150.1, 152.7, 164.2;
ESI-MS m/z: 604.3 [M+H]+, 626.1 [M+Na]+; Anal. Calcd For
5.8.9. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl) methoxy)phenyl)-4-
methylbenzamide (9)
Yield 38.0%; pale yellow powder; mp: 122–124 °C; 1H
NMR(300 MHz, DMSO-d6): d = 9.31 (s, 1H, CONH), 8.18 (s, 1H,
C32H37N5O7: C, 63.67; H, 6.18; N, 11.60. Found: C, 63.71; H, 6.19;
N, 11.65.
Please cite this article in press as: Jiao, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.032

L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
5.8.13. 4-Cyano-N-(2-((1-(2-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)benzamide (13)
75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.6, 109.8,
111.7, 113.9, 120.9, 122.4, 124.5, 125.2, 125.7, 126.0, 126.1,
126.3, 126.9, 130.2, 133.9, 136.0, 142.6, 146.9, 147.1, 147.7,
150.9, 163.1; ESI-MS m/z: 559.3 [M+H]+, 581.3 [M+Na]+; Anal.
Calcd For C29H30N6O6: C, 62.36; H, 5.41; N, 15.05. Found: C,
66.27; H, 5.40; N, 15.02.
Yield 63.2%; white powder; mp: 154–156 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.75 (s, 1H, CONH), 8.15 (s, 1H, NCH@C), 8.07 (d,
J = 8.2 Hz, 2H, ArH), 7.99 (d, J = 8.2 Hz, 2H, ArH), 7.71 (d,
J = 7.5 Hz, 1H, ArH), 7.30 (d, J = 8.0 Hz, 1H, ArH), 7.18 (dd,
J = 6.9 Hz, 6.9 Hz, 1H, ArH), 7.00 (dd, J = 7.4 Hz, 7.4 Hz, 1H, ArH),
6.61 (s, 1H, ArH), 6.56 (s, 1H, ArH), 5.23 (s, 2H, OCH2), 4.55 (t,
J = 5.9 Hz, 2H, CHNCH2), 3.68 (s, 3H, –OCH3), 3.67 (s, 3H, –OCH3),
3.50 (s, 2H, ArCH2N), 2.86 (t, J = 5.9 Hz, 2H, NCH2), 2.61 (s, 4H,
2 ꢀ CH2); 13C NMR (DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0,
54.8, 55.4, 56.6, 62.6, 109.8, 111.7, 113.8, 114.0, 118.3, 121.0,
124.5, 124.9, 125.7, 126.2, 127.0, 128.3, 132.5, 138.5, 142.6,
146.9, 147.2, 150.7, 163.7; ESI-MS m/z: 539.4 [M+H]+, 561.1
[M+Na]+; Anal. Calcd for C30H30N6O4: C, 66.90; H, 5.61; N, 15.60.
Found: C, 66.83; H, 5.63; N, 15.67.
5.8.17. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)
methoxy)phenyl)quinoline-2-carboxamide (17)
Yield 35.5%; white powder; mp: 179–184 °C; 1H NMR(300 MHz,
DMSO-d6): d = 10.84 (s, 1H, CONH), 8.64 (d, J = 8.4 Hz, 1H, ArH),
8.47 (d, J = 7.5 Hz, 1H, ArH), 8.36 (s, 1H, NCH@C), 8.26 (d,
J = 8.4 Hz, 1H, ArH), 8.12 (d, J = 8.4 Hz, 1H, ArH), 7.90 (s, 2H, ArH),
7.75 (m, 1H, ArH), 7.35 (d, J = 7.9 Hz, 1H, ArH), 7.09 (m, 2H, ArH),
6.60 (s, 1H, ArH), 6.54 (s, 1H, ArH), 5.36 (s, 2H, OCH2), 4.61 (t,
J = 5.5 Hz, 2H, CHNCH2), 3.66 (s, 3H, –OCH3), 3.64 (s, 3H, –OCH3),
3.47 (s, 2H, ArCH2N), 2.90 (t, J = 5.5 Hz, 2H, NCH2), 2.50 (s, 4H,
2 ꢀ CH2); 13C NMR (DMSO, 75 MHz, d ppm): 27.9, 47.2, 50.0,
54.8, 55.4, 56.7, 62.4, 109.7, 111.6, 112.7, 118.1, 118.6, 121.3,
124.1, 124.9, 125.6, 126.1, 127.3, 128.1, 128.5, 129.0, 130.8,
138.6, 142.2, 147.3, 149.2, 161.1; ESI-MS m/z: 565.3 [M+H]+,
587.1 [M+Na]+; Anal. Calcd for C32H32N6O4: C, 68.07; H, 5.71; N,
14.88. Found: C, 67.88; H, 5.73; N, 14.91.
5.8.14. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3,5-
dimethoxybenzamide (14)
Yield 69.8%; white powder; mp: 122–124 °C; 1H NMR
(300 MHz, DMSO-d6): d = 9.35 (s, 1H, CONH), 8.16 (s, 1H, NCH@C),
7.78 (d, J = 7.3 Hz, 1H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 7.15 (dd,
J = 7.1 Hz, 7.1 Hz, 1H, ArH), 7.06 (s, 2H, ArH), 6.99 (dd, J = 7.5 Hz,
7.5 Hz, 1H, ArH), 6.70 (s, 1H, ArH), 6.60 (s, 1H, ArH), 6.55 (s, 1H,
ArH), 5.22 (s, 2H, OCH2), 4.54 (t, J = 5.7 Hz, 2H, CHNCH2), 3.80 (s,
6H, 2 ꢀ –OCH3), 3.68 (s, 3H, –OCH3), 3.67(s, 3H, –OCH3), 3.49 (s,
2H, ArCH2N), 2.85 (t, J = 5.8 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2);
13C NMR (DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4,
56.6, 62.5, 103.6, 105.2, 109.8, 111.7, 113.7, 121.0, 124.0, 124.6,
125.5, 125.7, 126.2, 127.4, 136.6, 142.6, 146.9, 147.1, 150.1,160.4,
164.4; ESI-MS m/z: 574.4 [M+H]+, 596.7 [M+Na]+; Anal. Calcd for
5.8.18. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)quinoline-3-carboxamide (18)
Yield 30.1%; white powder; mp: 148–150 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.39 (s, 1H, CCHN), 9.01 (s, 1H, CONH), 8.25 (s, 1H,
ArH), 8.25 (s, 1H, NCH@C), 8.20 (m, 3H, ArH), 7.96 (ddd, J = 7.0 Hz,
7.0 Hz, 1.3 Hz, 1H, ArH), 7.85 (m, 2H, ArH), 7.38 (d, J = 7.3 Hz,
1H, ArH), 7.25 (ddd, J = 7.6 Hz, 7.6 Hz, 1.3 Hz, 1H, ArH), 6.61 (s,
1H, ArH), 6.55 (s, 1H, ArH), 5.32 (s, 2H, OCH2), 4.59 (t, J = 6.1 Hz,
2H, CHNCH2), 3.73 (s, 3H, –OCH3), 3.70 (s, 3H, –OCH3), 3.50 (s, 2H,
ArCH2N), 2.88 (t, J = 6.1 Hz, 2H, NCH2), 2.58 (m, 4H, 2 ꢀ CH2); 13C
NMR (DMSO, 75 MHz, d ppm): 27.9, 47.1, 49.9, 54.8, 55.4, 56.6,
62.8, 72.0,109.76, 111.7, 114.1, 121.1, 124.5, 124.6, 125.6, 126.0,
126.1, 126.5, 127.3, 127.5, 128.7, 129.2, 131.4, 135.9, 142.7, 146.8,
147.1, 148.5, 148.9, 150.6, 163.8; ESI-MS m/z: 565.4 [M+H]+,
587.3 [M+Na]+; Anal. Calcd for C32H32N6O4: C, 68.07; H, 5.71; N,
14.88. Found: C, 68.21; H, 5.73; N, 14.83.
C31H35N5O6: C, 64.91; H, 6.15; N, 12.21. Found: C, 64.83; H, 6.11;
N, 12.24.
5.8.15. 3-Chloro-N-(2-((1-(2-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)benzamide (15)
Yield 73.0%; pale yellow powder; mp: 150–152 °C; 1H
NMR(300 MHz, DMSO-d6): d = 9.54 (s, 1H, CONH), 8.15 (s, 1H,
NCH@C), 7.94 (d, J = 8.4 Hz, 2H, ArH), 7.74 (d, J = 7.5 Hz, 1H, ArH),
7.57 (d, J = 8.4 Hz, 2H, ArH), 7.29 (d, J = 8.0 Hz, 1H, ArH), 7.15 (dd,
J = 7.6 Hz, 7.6 Hz, 1H, ArH), 7.00 (dd, J = 7.6 Hz, 7.6 Hz, 1H, ArH),
6.61 (s, 1H, ArH), 6.56 (s, 1H, ArH), 5.23 (s, 2H, OCH2), 4.55 (t,
J = 5.9 Hz, 2H, CHNCH2), 3.68 (s, 3H, –OCH3), 3.67 (s, 3H, –OCH3),
3.50 (s, 2H, ArCH2N), 2.85 (t, J = 5.7 Hz, 2H, NCH2), 2.61 (s, 4H,
2 ꢀ CH2); 13C NMR (DMSO, 75 MHz, d ppm): 28.0, 47.1, 50.0,
54.8, 55.4, 56.6, 62.6, 109.8, 111.7, 114.0, 121.0, 124.5, 125.7,
125.8, 126.2, 127.4, 128.5, 129.4, 133.2, 136.4, 142.6, 146.9,
147.2, 150.4, 163.9; ESI-MS m/z: 548.4 [M+H]+, 570.4 [M+Na]+;
Anal. Calcd for C29H30ClN5O4: C, 63.56; H, 5.52; Cl, 6.47; N, 12.78.
Found: C, 63.62; H, 5.51; Cl, 6.50; N, 12.74.
5.8.19. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)nicotinamide (19)
Yield 58.3%; white powder; mp: 187–190 °C; 1H NMR(300 MHz,
DMSO-d6): d = 9.76 (s, 1H, CONH), 8.34 (d, J = 7.7 Hz, 1H, ArH), 8.22
(s, 1H, NCH@C), 7.79 (d, J = 6.9 Hz, 1H, ArH), 7.64 (m, 3H, ArH), 7.35
(d, J = 7.7 Hz, 1H, ArH), 7.23 (ddd, J = 8.0 Hz, 8.0 Hz, 1.3 Hz, 1H,
ArH)), 7.05 (dd, J = 7.3 Hz, 7.3 Hz, 1H, ArH), 6.67 (s, 1H, ArH), 6.61
(s, 1H, ArH), 5.29 (s, 2H, OCH2), 4.59 (t, J = 5.8 Hz, 2H, CHNCH2),
3.74 (s, 3H, –OCH3), 3.73 (s, 3H, –OCH3), 3.55 (s, 2H, ArCH2N),
2.91 (s, 2H, NCH2), 2.67 (s, 4H, 2 ꢀ CH2); 13C NMR (DMSO,
75 MHz, d ppm): 28.0, 47.1, 50.0, 54.8, 55.4, 56.6, 62.6, 109.8,
111.7, 113.9, 120.9, 124.6, 124.8, 125.7, 126.0, 126.2, 127.1,
134.9, 142.6, 146.9, 147.1, 150.6, 164.0; ESI-MS m/z: 515.4
[M+H]+, 537.4 [M+Na]+; Anal. Calcd for C28H30N6O4: C, 65.35; H,
5.88; N, 16.33. Found: C, 65.47; H, 5.83; N, 16.30.
5.8.16. N-(2-((1-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3-
nitrobenzamide (16)
Yield 53.8%; yellow powder; mp: 150–152 °C; 1H
NMR(300 MHz, DMSO-d6): d = 9.94 (s, 1H, CONH), 8.73 (s, 1H,
NCH@C), 8.40 (m, 2H, ArH), 8.18 (s, 1H, ArH), 7.80 (dd, J = 8.0 Hz,
8.0 Hz, 1H, ArH), 7.69 (d, J = 7.4 Hz, 1H, ArH), 7.31 (d, J = 7.9 Hz,
1H, ArH), 7.20 (dd, J = 8.0 Hz, 8.0 Hz, 1H, ArH), 7.01 (dd,
J = 7.3 Hz, 7.3 Hz, 1H, ArH), 6.60 (s, 1H, ArH), 6.54 (s, 1H, ArH),
5.23 (s, 2H, OCH2), 4.55 (t, J = 5.9 Hz, 2H, CHNCH2), 3.68 (s, 3H,
–OCH3), 3.67 (s, 3H, –OCH3), 3.49 (s, 2H, ArCH2N), 2.85 (t,
J = 5.6 Hz, 2H, NCH2), 2.61 (s, 4H, 2 ꢀ CH2); 13C NMR (DMSO,
5.9. Biological assays
5.9.1. MTT assay
K562/A02 cells were incubated in RPMI 1640 medium supple-
mented with 10% fetal bovine serum at 37 °C in a 5% CO2 humidi-
fied atmosphere. K562/A02 cells (1ꢁ2 ꢀ 104 cells per well) were
Please cite this article in press as: Jiao, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.032

10
L. Jiao et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
seeded in 96-well plates. After 24 h incubation, cells were treated
with various concentrations of ADM in absence or presence of tar-
get 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
Acknowledgments
The work was supported by the National Science and Technol-
ogy Major Projects of China (No. 2009ZX09102-033) and the
National Natural Science Foundation of China (No. 81173088).
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.
References and notes
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5.9.2. Duration of the MDR reversal
1ꢁ2 ꢀ 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 7, VRP or PBS at the con-
centration of 5 lM before being washed 0 or 3 times with growth
medium. Then, the cells were incubated for 0, 6 or 12 h before the
addition of various concentrations of ADM or vehicle. The incuba-
tion was continued for 48 h prior to the MTT analysis. The IC50 val-
ues of the compounds for cytotoxicity were calculated by
GraphPad Prism 5.0 software from the dose–response curves.
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5.9.3. Flow cytometry
5.9.3.1. Accumulation of Rh123 and ADM.
K562 and K562/
A02 cells were seeded into 24-well plates at 1.5 ꢀ 105/well. Differ-
ent concentrations of compound 7 and VRP were pre-incubated
with cells for 60 min. Then Rh123 or ADM were added into every
well and incubated for 90 min, washed three times with PBS at
4 °C for intracellular mean fluorescence intensity (MFI) analysis.
The mean fluorescence intensity of retained intracellular Rh123
was estimated by BD FACSCalibur flow cytometer. The ADM
accumulation level was showed in photographs taking by using
fluorescence microscope.
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5.9.4. Rhodamine123 efflux assay
K562 or K562/A02 cells were seeded into 24-well plates at 1.5
ꢀ105/well and incubated with 5
lM Rh123 for 60 min before
washing with ice PBS for three times. Then the cells were incu-
bated with or without various concentrations of compound 7 or
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VRP (0.5, 2.5, 5 lM) for another 90 min. Afterwards the cells were
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retained intracellular Rh123 was estimated by BD FACSCalibur
flow cytometer.
Please cite this article in press as: Jiao, L.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.032